JP2022502048A - Use of glutamine synthetase to treat fatty liver disease - Google Patents

Abstract

The present invention relates to the treatment of fatty liver disease by administration of glutamine synthetase.

Description

The present invention relates to the use of glutamine synthetase for the treatment of fatty liver disease. Pharmaceutical compositions containing glutamine synthetase are also provided.

A healthy liver typically contains no or little fat. Both early-stage alcoholic and non-alcoholic liver disease are characterized by the appearance of liver fat deposits. Alcohol Related Liver Disease (ARLD) due to excessive alcohol intake increases in severity to hepatitis, and cirrhosis if left untreated from the appearance of fat deposits. In the most severe cases, ARLD may progress to liver cancer. Similarly, non-alcoholic fatty liver disease (NAFLD) typically begins with an increase in fat in the liver of an overweight or obese person. NAFLD also has several levels of severity, from fatty liver deposits in the liver to fibrosis, scarring and cirrhosis of the liver. Currently, there is no treatment for fatty liver disease other than changes in alcohol intake and dietary and lifestyle changes.

Fatty liver disease is sometimes referred to as steatosis or simple steatosis. Fat accumulation may be accompanied by inflammation. This is a condition known as steatohepatitis, which is more progressive than simple fatty liver or simple steatoopathy.

NAFLD can be due to metabolic factors, some of which can have genetic causes such as glycogen storage disease, Weber-Christian disease and the like. Other causes include undernutrition, malnutrition, and the use of drugs or toxicants, including chemotherapy and antiretroviral therapy.

The pathophysiology of fatty liver disease is represented by the appearance of small fatty vacuoles around the nucleus of hepatocytes. As the size of the vacuole increases, the nucleus is pushed around the cell and the cell can exhibit an annular appearance. The vacuoles often appear empty because the fat dissolves during the processing of the tissue. Large, adherent vacuoles may form cysts that are considered irreversible.

Condition management may also include dietary control to limit protein intake and ensure adequate nutritional intake, including control of protein and / or nitrogen intake as well as parenteral intake of calories.

However, despite improved treatment and management of the condition, current therapies are non-specific and have not always been successful in managing the condition. Therefore, further or improved therapy for fatty liver disease continues to be needed.

The present invention attempts to address this need and is based on the concept of treating fatty liver disease using glutamine synthetase.

In particular, the present invention provides glutamine synthetase for use in treating fatty liver disease by reducing the amount of fat deposits in the liver of a subject. The glutamine synthetase may be provided to the subject as a nucleic acid molecule as a gene therapy. Alternatively, glutamine synthetase may be administered as protein therapy.

Glutamine synthetase (GS) catalyzes the reaction:
Glutamic acid + ATP + NH ₃ → Glutamine + ADP + phosphoric acid.

The present invention is based on the surprising observation that administration of glutamine synthetase improves the symptoms of fatty liver in hepatocytes. Specifically, glutamine synthetase has been shown to have the effect of reducing or eliminating fat deposition in hepatocytes.

The invention is also based on the surprising observation that administration of glutamine synthetase prevents the progression of fibrosis in liver tissue. Specifically, glutamine synthetase has been shown to have the effect of reducing or eliminating fibrosis in liver tissue.

Glutamine is an important amino acid with numerous functions including maintenance of intestinal permeability and immune function, both of which maintain intestinal permeability and immune function were observed in this unexpected manner of glutamine synthetase in the treatment of NAFLD. May contribute to action.

In addition, the combination of GS and an ammonia-lowering agent (such as a nitrogen scavenger, eg, a pharmaceutically acceptable salt of phenylacetic acid such as sodium phenylacetic acid) has a synergistic effect and reduces or eliminates fat deposition in the liver. The ability of GS can be further enhanced.

Accordingly, in one embodiment, the invention provides glutamine synthetase (GS) for use in the treatment of fatty liver disease by reducing or eliminating hepatic fat deposition. GS reduces or ameliorate the cellular effects of fatty liver disease such as hepatocyte or tissue fat deposition and therefore ameliorate or prevent the progression of fatty liver disease in liver tissue, especially simple fatty liver disease. Accordingly, the present invention provides GS for use in methods of treating or preventing fatty liver disease and related liver diseases including hepatitis, fibrosis, cirrhosis and cancer.

In a suitable embodiment, GS is provided for use in the treatment of fatty liver disease by reducing or eliminating hepatic fat deposits and may be provided for use in combination with an ammonia lowering agent.

In another aspect, the invention provides an ammonia-lowering agent for use in combination with GS for use in the treatment of fatty liver disease by reducing or eliminating hepatic fat deposition.

A relevant aspect of the invention is also the GS for the manufacture of compositions for the treatment of fatty liver disease by reducing or eliminating hepatic fat deposits (eg, pharmaceutical or nutritional compositions, such as pharmaceuticals or supplements). Also provides use.

In a suitable embodiment, the composition may be intended for use in combination with an ammonia lowering agent.

A further aspect of the invention also provides the use of an ammonia-lowering agent to produce a composition for use in combination with GS for the treatment of fatty liver disease by reducing or eliminating hepatic fat deposition.

In a further aspect, the invention provides the use of GS and ammonia lowering agents to produce compositions for use in the treatment of fatty liver disease by reducing or eliminating hepatic fat deposits.

In a further aspect, the present invention is a method of treating fatty liver disease in a subject by reducing or eliminating hepatic fat deposition, wherein GS is administered to the subject (more specifically, a subject in need thereof). Provide a method that includes that. In a suitable embodiment, the method further comprises administering an ammonia lowering agent. In a suitable embodiment, the method may include simultaneous, continuous or subsequent administration of glutamine synthetase (GS) and an ammonia lowering agent to said subject. The method of the present invention may include a step of diagnosing a subject having fatty liver disease. The methods of the invention may include monitoring subjects with fatty liver disease during and after treatment.

Also provided is a composition comprising GS for use in the treatment of fatty liver disease by reducing or eliminating hepatic fat deposition in a subject.

Appropriately, the composition comprising GS may be a pharmaceutical composition or a nutritional composition, such as a pharmaceutical or supplement. Appropriately, the composition may further comprise an ammonia lowering agent.

The present invention also relates to a composition comprising GS and an ammonia lowering agent.

Appropriately, the composition may be a pharmaceutical composition or a nutritional composition. Suitably, the composition may be for use in the treatment of fatty liver disease by reducing or eliminating hepatic fat deposits.

In another embodiment, the invention is used in an expression vector encoding a glutamine synthase or a biologically active fragment or variant thereof for use in the treatment of adipose liver disease, and in the treatment of adipose liver disease. To provide cells comprising an expression vector selected from the group consisting of an expression vector encoding a glutamine synthase or a biologically active fragment or variant thereof for use in combination with an ammonia lowering agent.

In another aspect, the invention provides a kit comprising an expression vector encoding a glutamine synthetase or a biologically active fragment or variant thereof, and an additional therapeutic agent. Appropriately, the additional therapeutic agent may be an agent that is effective against fatty liver disease. Suitably, the agent effective against fatty liver disease is an ammonia lowering agent or an amino acid thereof or a urea cycle intermediate. The kit may be provided for use in methods of treating fatty liver disease.

In another aspect, the invention provides a product comprising GS and additional therapeutic agents as a combination preparation for individual, simultaneous or continuous use in the treatment of fatty liver disease. Appropriately, the additional therapeutic agent may be an agent that is effective against fatty liver disease. Suitable agents that are effective against hyperammonemia are ammonia lowering agents or their amino acids or urea cycle intermediates. More appropriately, a further therapeutic agent is a nitrogen scavenger.

In another aspect, the invention provides glutamine synthetase (GS) for use in the treatment of late fatty liver disease by reducing or eliminating fibrosis of the liver. Therefore, GS improves or prevents the progression of fatty liver disease, especially advanced or late fatty liver disease and cirrhosis. Accordingly, the present invention provides GS for use in methods of treating or preventing late fatty liver disease, including hepatitis, fibrosis, cirrhosis and cancer, by reducing or eliminating liver fibrosis.

In a suitable embodiment, the composition may be intended for use in combination with an ammonia lowering agent.

In another aspect, the invention provides an ammonia-lowering agent for use in combination with GS for use in the treatment of fatty liver disease by reducing or eliminating liver fibrosis.

A relevant aspect of the invention is also the GS for the manufacture of compositions for the treatment of fatty liver disease by reducing or eliminating fibrosis of the liver (eg, pharmaceutical or nutritional compositions, such as pharmaceuticals or supplements). Provide use.

In appropriate embodiments, compositions for use in combination with an ammonia-lowering agent for the treatment of fatty liver disease by reducing or eliminating liver fibrosis (eg, pharmaceutical or nutritional compositions, such as pharmaceuticals or supplements). ) May be provided for the use of GS. The composition may be for combined use, simultaneous, individual or continuous administration of GS and an ammonia lowering agent.

A further aspect of the invention also provides the use of an ammonia-lowering agent for the manufacture of compositions for use in combination with GS for the treatment of fatty liver disease by reducing or eliminating liver fibrosis.

In a further aspect, the invention provides the use of GS and ammonia lowering agents for the manufacture of compositions for use in the treatment of fatty liver disease by reducing or eliminating liver fibrosis.

In a further aspect, the invention is a method of treating fatty liver disease in a subject by reducing or eliminating fibrosis of the liver, wherein the subject (more specifically, the subject in need thereof) is administered GS. Provide a method that includes that. In a suitable embodiment, the method further comprises administering an ammonia lowering agent. In a suitable embodiment, the method may include simultaneous, continuous or subsequent administration of glutamine synthetase (GS) and an ammonia lowering agent to said subject. The method of the present invention may include a step of diagnosing a subject having fatty liver disease. The methods of the invention may include monitoring subjects with fatty liver disease during and after treatment.

Also provided is a composition comprising GS for use in the treatment of fatty liver disease for a subject by reducing or eliminating liver fibrosis.

Appropriately, the composition comprising GS may be a pharmaceutical composition or a nutritional composition, such as a pharmaceutical or supplement. Appropriately, the composition may further comprise an ammonia lowering agent.

The present invention also relates to a composition comprising a GS and an ammonia lowering agent.

Appropriately, the composition may be a pharmaceutical composition or a nutritional composition. Suitably, the composition may be for use in the treatment of fatty liver disease by reducing or eliminating fibrosis of the liver.

In another embodiment, the invention is an expression vector encoding a glutamine synthase or a biologically active fragment or variant thereof for use in the treatment of fatty liver disease, and reduction or elimination of liver fibrosis. Selected from the group consisting of an expression vector encoding a glutamine synthase or a biologically active fragment or variant thereof for use in combination with an ammonia lowering agent for use in the treatment of fatty liver disease. A cell containing an expression vector is provided.

In another embodiment, the kit as defined herein may be provided for use in a method of treating fatty liver disease by reducing or eliminating fibrosis of the liver.

In another aspect, the invention provides a product comprising GS and a further therapeutic agent as a combination preparation for individual, simultaneous or continuous use in the treatment of fatty liver disease by reducing or eliminating liver fibrosis. .. Appropriately, the additional therapeutic agent may be an agent that is effective against fatty liver disease. Suitable agents that are effective against hyperammonemia are ammonia lowering agents or their amino acids or urea cycle intermediates. More appropriately, a further therapeutic agent is a nitrogen scavenger.

The present invention may be used in the treatment of subjects with acquired or hereditary fatty liver disease. The subject may have a genetic (inherited) fatty liver disease associated with urea cycle dysfunction (UCD). The subject may have a hereditary disorder due to a deficiency of the urea cycle enzyme or transporter. Accordingly, the present invention relates to the treatment of a subject with adipose liver disease, wherein the subject is a UCD, or other genetically acquired hyperammonemia disease, preferably a deficiency of ornithine transcarbamilas (ie, protein abnormality). Ornithine transcarbamylase gene mutations or genetic abnormalities) that result in different expression. Subjects with UCD or other genetic hyperammonemia may exhibit symptoms of fatty liver disease, such as hepatocyte fat deposition or liver tissue fibrosis. Subjects may have hereditary disorders associated with fatty liver disease, such as glycogen storage disorders or Christian Weber disease. A subject may have acquired fatty liver disease and may have one or more conditions selected from the group consisting of diabetes, obesity, malnutrition, excessive alcohol intake, and substance use. The subject may have one of the above conditions and is diagnosed at risk of developing fatty liver disease.

The invention described herein also relates to treating liver tissue removed from a subject, for example, with an ex vivo treatment method.

The term "GS" may include either a GS protein or a nucleic acid sequence encoding a GS protein or an active fragment thereof. As used herein, GS includes any biologically active fragment or variant of a protein or nucleic acid sequence within its scope. The term "GS protein" can also be expressed as "protein with glutamine synthetase (GS) activity". The term "protein" is widely used herein to include peptides and polypeptides, as well as any proteinaceous molecule, including proteins or polypeptide fragments. As described in more detail below, a GS protein may or may not be a full-length GS enzyme when it appears naturally (eg, natural or wild-type GS), or does not correspond to the GS enzyme. Well, cleaved or other variants are included. Also included are conjugates or fusions of GS proteins with other molecules, as described in more detail below.

The term "fatty liver disease" includes any condition in which fatty deposits are present in hepatocytes. Such conditions include hereditary or acquired fatty liver disease (fatty liver disease) including ARLD and NAFLD, and liver inflammation (fatty hepatitis including NASH), hepatitis (alcoholic and non-alcoholic), cirrhosis, Conditions derived from fatty liver diseases such as fibrosis and liver cancer can be mentioned. Aspects of the invention can be used to reduce or eliminate hepatic fat deposition and / or to completely or partially improve hepatic fibrosis or scarring. Aspects of the invention also inhibit or improve the progression of fatty liver disease, eg, delay or improve the progression of the disease from simple fatty liver to steatohepatitis (including NASH), fibrosis, cirrhosis, or liver cancer. It can also be used to prevent. Aspects of the invention relate to the treatment or prevention of disease by improving the symptoms of the disease, eg, improving the symptoms of fatty liver and / or fibrosis. Accordingly, aspects of the invention relate to reducing or eliminating hepatic fat deposition and / or scarring. Accordingly, aspects of the invention relate to the treatment or prevention of fatty liver disease and related liver diseases including hepatitis, fibrosis, cirrhosis and cancer.

In fatty liver disease, elevated ammonia levels can be observed. Therefore, plasma (or blood) ammonia concentrations of> 40, 60 or 70 or 80 μmol / L or higher, such as 41, 42, 45, 50, 55, 60, 70 or 80 μmol / L or higher, can be observed. .. For example, plasma ammonia concentrations> 100 μmol / L associated with normal anion gaps and normal plasma glucose concentrations, and especially plasma ammonia concentrations above 150 μmol / L, are fatty liver, eg, due to changes in urea cycle enzyme (UCE) expression and / or activity. It may or may not be associated with the disease.

Fatty liver disease is a blood test for liver enzymes such as alanine aminotransferase and aspartate aminotransferase above normal levels by imaging methods such as ultrasound, CT scan, or MRI; techniques well known and used in the art. Measurement of collagen proportionate area of liver tissue by staining (eg, plasma or serum or any blood-derived sample), or detection of symptoms such as fatty deposition or fibrosis or scarring of liver or liver tissue Can be detected using a liver biopsy for. Such decisions and analyzes can be combined with clinical evaluations such as liver function tests. Family history studies and / or molecular genetic testing and / or evaluation may also be performed. Accordingly, the invention may include diagnosing a subject for fatty liver disease, symptoms of hyperammonemia, or a hereditary disorder resulting in fatty liver (eg, UCD) or hyperammonemia. In another aspect of the invention, the subject has a hereditary disorder that has or results in fatty liver disease (eg, UCD), or a condition that poses a higher risk of fatty liver disease, such as diabetes, obesity, malnutrition, etc. Those who have been diagnosed with excessive alcohol intake or drug use.

As used herein, the internal name AM-535 refers to glutamine synthetase. AM-535 may include human GS having one or more N-terminal linkers and may have a poly (his) tag. AM-535 may include a pegged version of GS. AM-535 is further described in the Examples. As used herein, references to glutamine synthetases or GS proteins for use according to the invention refer to human GS and non-human animals (mouse, bovine, rabbit, rat, monkey, chimpanzee, and dog, etc.). Includes references to all forms of enzymatically active GS, including GS derived from (such as) or from other sources including, for example fungi, plants or bacteria, as well as enzymatically active variants. Representative GS proteins are therefore the GS polypeptide set forth in SEQ ID NO: 1 or 2 or 4 (human GS, precursor, mature N-terminal tagged form, respectively), or SEQ ID NO: 6 [Lactobacillus acidophilus (Lactobacillus acidophilus). A GS polypeptide from Lactobacillus acididophilus strain 30SC] or SEQ ID NO: 7 [GS from corn, Zea Mays], or an enzymatically active fragment thereof, at least 50%, 55. %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or Those having higher sequence identity can be mentioned. For example, references to GS also include N and / or C-terminal cleaved polypeptides or amino acid modified proteins (eg, post-translational modifications such as adenylation, or amino acid polymorphisms that can affect the structure or activity of the protein. Modification) can also be included. References to GS can also include protein multimers. The term "GS protein" is therefore synthetic with all native GS enzymes or polypeptides, as well as one or more amino acid substitutions, additions (including insertions and extensions) or deletions that retain GS enzyme activity. Includes an enzymatically active fragment or variant thereof, including an induced and modified polypeptide.

The GS may therefore be any enzyme within the range of the enzyme classification EC 6.3.1.2, or may be derived from that enzyme. The GS may be any polypeptide or peptide having GS activity. GS activity can be defined as the ability to convert glutamic acid and ammonia to glutamine, eg, according to the reaction scheme shown above. GS activity can be assessed or determined using assays or tests known in the art and described in the literature (eg, functional activity assays). For example, the GS enzyme activity assay is described in Listrom et al, Biochem. J. 1997, 328, 159-163. Assays for GS activity are also described in the following examples (see Examples 2 and 4).

Human GS is expressed as a polypeptide of 373 amino acids (shown in SEQ ID NO: 1). This corresponds to a complete "precursor" protein when expressed, which protein is then further processed into a mature form with amino acids 2-373 (maturation of the 372 amino acids set forth in SEQ ID NO: 2). Only the N-terminal methionine has been removed to bring the protein in vivo. The exemplary polynucleotide set forth in SEQ ID NO: 3 corresponds to the cDNA encoding the polypeptide of SEQ ID NO: 1. No. 4 corresponds to a modified human GS protein comprising the GS polypeptide of SEQ ID NO: 1 with an N-terminal His tag and linker sequence, as prepared and used in the following examples. Is a cDNA sequence encoding the polypeptide of SEQ ID NO: 4, codon-optimized for expression in bacteria, as used in the following examples.

Human GS is well characterized (eg Listrom et al., 1997, see above). GS enzymes from other organisms, including plants and bacteria, have also been identified, and the nucleic acid and amino acid sequences of such other GS enzymes are well known in the art, eg, the National Center for Biotechnology Information (NCBI). It is provided in freely available databases such as nucleotide (ncbi.nlm.nih.gov/nuccore) and protein (ncbi.nlm.nih.gov/protein) databases. Sequence identity between plant or bacterial GS enzymes and human GS can be low, but structural and functional similarities are high. Therefore, plant or bacterial GS, or actually GS from other organisms, or amino acid sequence variants thereof can be used. As a representative example, SEQ ID NO: 6 is a Lactobacillus acidophilus strain having 23.8% sequence identity with human GS and 61.9% sequence identity with Lactobacillus casei GS. The amino acid sequence of GS derived from 30SC is described, and SEQ ID NO: 7 is a GS amino acid derived from corn (corn, Zea-Mice) having 55.7% sequence identity with human GS. Describe the sequence.

GS generally occurs as a multimer containing multiple (ie, two or more) monomer subunits. For example, the GS amino acid sequence provided above corresponds to such a monomeric subunit. Human GS is most frequently reported as a pentamer or decamer (5 or 10 subunits). As used herein, GS may be provided as a monomer and / or a multimer. The multimer may contain two or more monomeric subunits, such as 2-20, 2-16, 2-15, 2-14 or 2-12 subunits. Recombinant GS can be present as a pentamer, decamer, or other multimer, or active monomer.

The GS protein used in the methods provided herein is the art of recombinant methods, protein isolation and purification methods, and chemical synthesis methods, provided that the resulting GS exhibits enzymatic activity. It can be obtained by any method known in. Thus, the GS may be recombinant GS, natural GS isolated from tissue, or chemically synthesized GS.

Modification of a GS polypeptide, such as the polypeptide set forth in SEQ ID NO: 1, to generate an enzymatically active GS variant for use in the methods provided herein is sufficient to those of skill in the art. Is within the range of the ability of. For example, one of ordinary skill in the art will appreciate that modifications at positions involved in substrate binding, or active sites, may be less tolerable than modifications at positions outside these important regions. Any GS polypeptide can be tested using methods well known in the art, such as those described in the art.

The GS polypeptide may be operably linked to one or more linkers and / or tag sequences. The linker sequence may be any suitable peptide sequence of 1 to 30 amino acids, more preferably 1 to 20 amino acids, more preferably 1 to 10 amino acids. The most suitable linker sequence may be 3, 4, 5, 6, 7, 8, 9 or 10 amino acid lengths. Appropriately, the linker sequence may contain glycine and / or serine residues that provide flexibility. The most suitable linker may be GGGS or GGGGS. The GS may be operably linked to two or more linkers, which may be the same or different. Suitably, one or more linkers may be operably linked (directly or indirectly via another peptide sequence) to the N-terminus of the polypeptide. When two or more linkers are provided, any one linker sequence can be operably directly linked to another linker or indirectly to another vector via an intervening sequence. The tag sequence may be any peptide sequence that appropriately binds to a matrix for protein isolation. Suitable tags are known and available in the art. Suitable tags include, for example, chitin-binding protein (CBP), maltose-binding protein (MBP), Strept tag and glutathione S-transferase (GST), and poly (His) tag. The poly (his) tag may be HHHHHH, or any suitable number of His residues. In a suitable embodiment, the GS sequence used in the present invention can be provided in the following conformation: linker-tag-linker-GS. Most appropriately, the linker may be as defined above, or more preferably GGGS or GGGGS. The tag may be as defined above, and most preferably a poly (his) tag. Most appropriately, the GS sequence may be provided as GGGS-poly (his) -GGGGS-GS. Suitably, the GS sequence is provided as GGGS-poly (his) -GGGGS-human GS. Appropriately, human GS is SEQ ID NO: 1. Also provided are GS proteins as defined herein and nucleic acid sequences encoding any linker and / or tag sequence. Such nucleic acid sequences may be provided in the vectors described herein.

When GS is provided as a nucleic acid molecule, GS may be a full-length native sequence encoding GS, eg, the sequence set forth in SEQ ID NO: 2, or in a biologically active fragment or variant thereof. There may be. The term "biologically active" refers to a fragment or variant of a nucleic acid encoding a glutamine synthetase (SEQ ID NO: 1) that exhibits the ability to convert glutamic acid to glutamine. The protein set forth in SEQ ID NO: 1 is encoded by the nucleic acid sequence set forth in SEQ ID NO: 2. Nucleic acid molecules encoding GS or biologically active fragments or variants thereof may be provided in vectors. The vector may be an expression vector.

The vector may include a fragment or variant of SEQ ID NO: 2 that encodes a biologically active fragment or variant of glutamine synthetase.

In a suitable embodiment, the vector further encodes an ammonia lowering agent.

As used herein, the term "variant" refers to a polypeptide comprising a modification of the primary structure of the polypeptide of SEQ ID NO: 1. Appropriately, the variant has 70% or more identity with the polypeptide of SEQ ID NO: 1; 80% or more identity with the polypeptide of SEQ ID NO: 1; 90% or more identity with the polypeptide of SEQ ID NO: 1; 95% or more identity with the polypeptide of SEQ ID NO: 1; 96% or more identity with the polypeptide of SEQ ID NO: 1; 97% or more identity with the polypeptide of SEQ ID NO: 1; 98 with the polypeptide of SEQ ID NO: 1 % Or more identity; or even 99% or more identity with the polypeptide of SEQ ID NO: 1 may be shared. The mutant is 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 10% or more, 20% or more, or more than the polypeptide of SEQ ID NO: 1 with respect to the sequence set forth in SEQ ID NO: 1. May differ by 30% or more.

As used herein, the term "fragment" refers to a polypeptide comprising a change in the length of the primary structure of the polypeptide of SEQ ID NO: 1. Suitable fragments may comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the full length of SEQ ID NO: 1. In fact, suitable variants may comprise at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 1.

In a suitable embodiment, the expression vector may be viral or non-viral. As an example, a suitable viral expression vector may be derived from a virus selected from the group consisting of paramixovirus, retrovirus, adenovirus, lentivirus, poxvirus, alphavirus, and herpesvirus. Other suitable viral vectors will be known to those of skill in the art.

Suitable non-viral expression vectors include inorganic particle expression vectors (such as calcium phosphate, silica, and gold), lipid-based particle expression vectors (eg, cationic lipids, lipid nanoemulches, and solid lipid nanoparticles) and polymer-based. May be selected from the group consisting of particle expression vectors [eg, peptides, polyethyleneimines, chitosans, and dendrimers]. Other suitable non-viral expression vectors will be known to those of skill in the art.

The term also includes a prodrug of a GS protein in a form that does not exhibit GS activity by itself but can be converted to active GS when administered to a subject.

Thus, as used herein, "enzymatically active" with respect to a GS protein or polypeptide refers to a GS protein or polypeptide that can catalyze the conversion of glutamic acid and ammonia to glutamine. Typically, the enzymatically active GS protein or polypeptide is at least or about 30%, 40%, 50% of the enzymatic activity of the GS polypeptide set forth in SEQ ID NO: 1, 2, 4, 6 or 7. , 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%.

As used herein, the term "subject" includes any human or non-human animal, such as humans, primates, livestock animals (eg sheep, pigs, cows, horses, donkeys), laboratory test animals. Refers specifically to mammals, including companion animals (eg dogs, cats) and captive wildlife (eg foxes, kangaroos, deer) (eg mice, rabbits, rats, guinea pigs). Preferably, the mammal is a human or laboratory test animal. Even more preferably, the mammal is a human.

As used herein, the terms "treating," "treatment," "preventing," and "prevention" are conditions or conditions that cure or ameliorate a condition or condition. Refers to any use that prevents the establishment of a disease, or otherwise prevents, prevents, delays, reduces or improves the progression of a condition or disease or other undesired condition in any way. Specifically, in the present invention, "treating" means reducing or eliminating fat deposition in the liver and completely or partially improving the symptoms of fatty liver disease. In this way, the triglyceride vacuoles present in the liver shrink or are eliminated. Progressive signs of liver disease, such as inflammation, fibrosis or scarring, can also be completely or partially improved. Therefore, the terms "treating" and "preventing" etc. should be considered in their broadest context. For example, treatment does not necessarily imply treatment until the patient is fully recovered, but includes any improvement or amelioration of the patient's or subject's condition, or the symptoms of the disease or condition. Thus, for example, in the case of hereditary fatty liver disease rather than acquired, the treatment according to the invention does not, of course, treat the underlying hereditary disorder, but treats the clinical condition that results from the fatty liver disease. In conditions that present or are characterized by multiple symptoms, treatment or prevention does not necessarily cure, improve, prevent, interfere, delay, reduce or improve all of the symptoms, but said. It may cure, improve, prevent, prevent, delay, reduce or improve one or more of the symptoms. In a suitable embodiment, the "treatment" according to the invention is a reduction of fat levels in the liver, eg, to normal or healthy levels, eg, fat is absent or very minimal fat vacuoles are present; fat. Absence of cysts; normal, non-displaced cell nuclei, resulting in no fibrosis or minimal / healthy levels of fibrosis. Therefore, treatment involves restoration of normal or healthy liver fat levels. Treatment may also include reduction or elimination of liver tissue fibrosis.

In a suitable embodiment, the "treatment" according to the invention may include an increase in glutamine synthetase levels and / or activity in the tissue of interest. Such an increase may be in any suitable tissue (eg, liver). Increased glutamine synthetase levels and / or activity can be determined, for example, by measuring glutamine levels, glutamate levels, and / or determining the ratio of glutamine levels to glutamate levels in a subject. .. The increase may be at normal or healthy levels, for example with fatty liver disease accompanied by a reduction in UCE. It will be appreciated that normal or healthy levels of glutamine and / or glutamic acid can vary depending on the sample in which they are measured. It will also be appreciated that normal or healthy glutamine and / or glutamate levels are subject-specific and may depend on factors such as subject weight, diet, gender and age. Normal or healthy levels of glutamine and / or glutamic acid will be known to those of skill in the art.

As used herein, "amelioration" refers to a reduction in the severity of at least one indicator or symptom of a condition or disease. In certain embodiments, improvement comprises delaying or slowing the progression of one or more indicators of a condition or disease. The severity of the indicator can be determined by subjective or objective measures known to those of skill in the art.

As used herein, the term "associated with" as used in the context of a disease or condition that is "related" to hepatic fat deposits means that the disease or condition is elevated in ammonia levels. It means that it can result from an increase in ammonia levels, can be characterized by an increase in ammonia levels, or can otherwise be associated with an increase in ammonia levels. Therefore, the association between disease or condition and fatty liver may be direct or indirect, or may be temporally separated.

Similarly, suitable samples for determining glutamine synthetase levels and / or glutamine synthetase activity include any suitable or desirable sample in which glutamine synthetase, glutamine and / or glutamic acid may be present. These may be any suitable or desirable tissue or body fluid sample. Examples of suitable tissues are liver and / or muscle tissue. Conveniently, the sample may be any body fluid sample, typically blood or any blood-derived sample, such as plasma or serum, but any other body fluid, such as urine, cerebrospinal. It may be a liquid, a feces or a tissue sample, for example, a biopsy sample or a cleaning fluid or a washing fluid sample. This can, of course, depend on the very nature of the condition, such as being treated.

As used herein, the term "effective amount" means a non-toxic but sufficient amount or dose of GS and / or ammonia lowering agent, depending on the context, to provide the desired effect. Included within the range of. It will be appreciated that the effective amounts of the protein and / or ammonia lowering agent may vary. Exemplary therapeutically effective amounts are specified elsewhere herein.

The exact amount or dose required will be per subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the specific GS being administered, and the mode of administration. Will be different. Therefore, it is not appropriate to specify the exact "effective amount". However, for any given case, a suitable "effective amount" can be determined by one of ordinary skill in the art using only routine experiments.

Indeed, a surprising feature of the invention is that, contrary to the literature reports of 5 or 10 subunit multimers, human GS is expressed as a mixture of multiple different types of multimers, including monomeric forms and / or. What you can get. The monomeric form has been shown to be active. Therefore, according to the invention, the GS can be used as a monomer and / or as a multimer, the multimer may be as a single multimer form or may or may not contain a monomer. It can be provided as a mixture of different multimer types. As reported in the examples below, 4 or more, eg 4-10, eg 5-8 multimeric forms can be obtained. The size of the multimer can be 2 to 20 subunits.

The following examples are for assessing the ability of the GS polypeptide to catalyze the conversion of glutamic acid and ammonia to glutamine.

In some examples, the GS protein used herein is a recombinant GS made using a prokaryotic or eukaryotic expression system well known in the art. Exemplary prokaryotic expression systems include, but are not limited to, Escherichia coli expression systems, and exemplary eukaryotic expression systems are limited to: Not, but include yeast, insect cell and mammalian cell expression systems.

Nucleic acids encoding GS can be obtained by any suitable method, including but not limited to RT-PCR of liver RNA and synthetic nucleotide synthesis. Primers for amplification can be designed based on known GS sequences such as those described above. The nucleic acid and amino acid sequences of GS are well known in the art, such as the National Center for Biotechnology Information (NCBI) nucleotides (ncbi.nlm.nih.gov/nuccore) and proteins (ncbi.nlm.nih.gov/protein). It is provided in freely available databases such as databases.

Nucleic acids encoding GS polypeptides, such as the nucleic acids having the sequence set forth in SEQ ID NO: 3, can be cloned into an expression vector suitable for the optimal expression system. In some examples, the nucleic acid is codon-optimized for expression in a particular system. For example, the nucleic acid encoding the GS polypeptide may be codon-optimized for expression in Escherichia coli. An exemplary codon-optimized nucleic acid encoding GS for expression in Escherichia coli is set forth in SEQ ID NO: 5, a His tag linked via a GGGGS linker (described in SEQ ID NO: 4). Encodes a GS polypeptide containing.

Typically, the nucleic acid encoding GS is cloned into an expression vector operably linked to a control sequence that promotes expression of a heterologous nucleic acid molecule. Many expression vectors suitable for GS expression are available and are known to those of skill in the art. The choice of expression vector is influenced by the choice of host expression system. Such choices are well within the level of skill of one of ordinary skill in the art. In general, expression vectors can include transcriptional promoters and optionally enhancers, translational signals, and transcriptional and translational termination signals. Expression vectors used for stable transformation typically have selectable markers that allow selection and maintenance of transformed cells. In some cases, an origin of replication can be used to amplify the number of copies of the vector in the cell.

The GS polypeptide can also be expressed as a protein fusion. For example, the fusion may be generated to add additional functionality to the polypeptide. Examples of fusion proteins include, but are not limited to, GS and affinity tags for purification (eg His tags such as his6, MYC, FLAG, HA or GST tags), leader sequences (pelB leader sequences and the like). , A sequence that directs protein secretion, or a protein that stabilizes and / or solubilizes GS (eg, maltose-binding protein (MBP)), or a protein that increases in vivo half-life (eg, albumin or Fc domain, or them). Fragments of) are included.

Prokaryotes, especially Escherichia coli, provide a system for making large quantities of GS. Transformation of Escherichia coli is a simple and rapid technique well known to those of skill in the art. The expression vector of Escherichia coli can contain an inducible promoter useful for inducing high levels of protein expression and for expressing proteins that are somewhat toxic to the host cell. Examples of inducible promoters include lac promoters, trp promoters, hybrid tac promoters, T7 and SP6 RNA promoters, and temperature controlled λPL promoters.

In another example, a eukaryotic expression system, such as a baculovirus expression system, is used to make the GS. Typically, the expression vector uses a promoter, such as the baculovirus polyhedrin promoter, for high level expression. Commonly used baculovirus systems include baculoviruses such as Autographa californica nuclear polyhedrosis virus (AcNPV) and silk moth (Bombyx mori) nuclear polyhedrosis virus (BmNPV). Sf9 from Spodoptera flugiperda, Pseudaletia unipuncata (A7S) and Danaus plexippus (Dpn) insect strains such as Danus plexippus (DpN). For high levels of expression, the GS nucleotide sequence is fused just downstream of the viral polyhedrin initiation codon.

Saccharomyces cerevisiae, Saccharomyces ponbe, Saccharomyces pombe, Yarrowia lipolytica, Yarrowia lipolytica, Kluyberomyces lypsis It can be used as an expression host for GS. Yeast can be transformed using an episomal replication vector or by stable chromosomal integration by homologous recombination. Typically, inducible promoters such as GALL, GAL7, and GAL5 are used to control gene expression. Yeast expression vectors often contain selectable markers such as LEU2, TRPl, HIS3, and URA3 for selecting and maintaining transformed DNA.

Mammalian expression systems can also be used to express GS. The expression construct can be introduced into mammalian cells by viral infection such as adenovirus, or by direct DNA introduction such as liposomes, calcium phosphate, DEAE-dextran, and physical means such as electrical perforation and microinjection. Expression vectors for mammalian cells typically include mRNA cap sites, TATA boxes, translation initiation sequences (Kozak consensus sequences) and polyadenylation elements. Such vectors often include transcriptional promoter-enhancers for high levels of expression, such as the SV40 promoter-enhancer, the human cytomegalovirus (CMV) promoter, and long-term repeats of Rous sarcoma virus (RSV). Exemplary cell lines available for mammalian expression include, but are not limited to, BHK, 293-F, CHO, Balb / 3T3, HeLa, MT2, mouse NSO (non-secreting) and others. Mice, rats, humans, monkeys, and chickens such as myeloma cell lines, hybridoma and heterohybridoma cell lines, lymphocytes, fibroblasts, Sp2 / 0, COS, NIH3T3, HEK293, 293S, 293T, 2B8, and HKB cells. And hamster cells.

After expression, GS includes, but is not limited to, SDS-PAGE, size fraction and size exclusion chromatography, ammonium sulfate precipitation, chelate chromatography, ion exchange chromatography and affinity chromatography to those skilled in the art. It can be purified using any known method. Affinity purification methods can be used to improve the efficiency and purity of the preparation. For example, antibodies and other molecules that bind GS can be used in affinity purification. As discussed above, the expression construct may be engineered to add affinity tags such as his, myc, FLAG or HA tags or GST moieties to the GS, followed by Ni resin, myc antibody, HA antibody, respectively. Affinity purification may be performed using FLAG antibody or glutathione resin. Suitably, a poly 9 × his tag may be used. More preferably, the 6 × his tag is used. Purity can be assessed by any method known in the art, including gel electrophoresis and staining and spectrophotometry such as SDS page and size exclusion chromatography (SEC).

The affinity tag (eg, his tag, etc.) may be removed for use according to the invention, but this is not always necessary and the GS polypeptide may be used with the attached tag.

The tag or other fusion partner may be attached to the GS via a linker, which may be any suitable linker, according to principles well known in the art. Such a linker may typically and conveniently be a short (eg 2-10, 2-8 or 2-6 mer) peptide. Examples may include linker GGSG, GGGS or GGGGS, but may consist of any suitable amino acid. Amino acid linkers allow the preparation of fusion proteins by recombinant means, but non-amino acid based linkers can also be used according to the principles and techniques well known in the art and described in the literature. The linker may be cleavable (eg enzymatically) or non-cleavable.

The GS polypeptide can be prepared as a bare polypeptide chain or as a modified polypeptide modified by binding or conjugating to additional moieties or chemical groups or substances. Exemplary modifications include, but are not limited to, pegging, albumination or other known modifications. For example, in some examples, the GS polypeptide for use in the described method is pegged using standard methods well known in the art. This can help increase the half-life of the circulating GS protein, for example. Therefore, in a preferred embodiment of the invention, the GS protein may be provided as a conjugate with a polymer such as polyethylene glycol (PEG) or polysaccharide or oligosaccharide. Conjugation with PEG is particularly preferred. As indicated above, the preparation of such conjugates is well known in the art and described in the literature. Therefore, various conjugations such as 100 daltons to 100 kD, but more often 5 kD to 100 kD, such as 12 or 15 kD to 60 or 80 kD, such as 15 to 50, 15 to 40, or 15 to 30 kD. Size PEG can be used. In addition, PEG can be attached or linked to GS proteins in various ways, and more than one PEG can be attached to each protein. The PEG can be linked directly or indirectly with respect to the fusion proteins described above, eg, via the linkers described, or by any molecular or chemical group capable of providing linker function. Thus, PEG may be one or both of the N or C ends, or the amino group of one or more lysine residues inside the GS molecule, eg, one or more lysine residues of the GS protein molecule, or any other chemical portion of the protein molecule or Can be linked by residues. Methods of binding or conjugating polymers such as PEG to proteins are well known in the art and described in the literature (eg, Roberts et al. 2012, Advanced Drug Delivery Reviews, 64 (supplement) 116-127). And Veronese 2001, Biomaterials 22, 405-417). The data presented in the examples below show that PEG conjugates prepared by ligating PEG to the N-terminus are particularly useful in, for example, in assaying the activity of liver lysates from animals to which various conjugates have been administered. It shows that there is. Therefore, a PEG conjugate containing PEG linked to the N-terminus of the GS protein is one preferred embodiment of the invention. The GS protein may be pegged in monomeric and / or multimeric form. Therefore, for convenience, preparations containing both monomeric and various multimer GS may be subjected to pegging.

The GS can be formulated as a pharmaceutical composition for administration to a subject. The GS can be formulated in any conventional manner by mixing selected amounts of GS with one or more physiologically or pharmaceutically acceptable carriers or excipients.

Accordingly, a further aspect of the invention provides a pharmaceutical composition comprising GS and one or more pharmaceutically acceptable carriers or excipients for the treatment of fatty liver disease by reducing hepatic fat deposition. do. The GS may be provided as the nucleic acid molecule or protein described herein.

The choice of carrier or excipient is within the skill of the dosing specialist and may depend on several parameters such as mode of dosing. In some examples, the GS protein is provided as a liquid. In another example, the GS protein is provided in a dried form, such as a desiccated or lyophilized form. Such dry forms can be rehydrated prior to administration by the addition of a suitable solution such as water, buffer, saline or other suitable solution. If the GS is provided as a nucleic acid, the GS may be provided in any suitable composition or may be injectable for direct administration to the liver. The GS provided herein may be formulated for direct administration, or for dilution or other modification. Therefore, the GS can be formulated in a single (or unit) or multiple dose form. Examples of single dose forms include ampoules and syringes. Examples of multiple dose forms include vials and bottles containing multiple unit doses.

The concentration of GS in the formulation, when administered, reduces or eliminates hepatic fat deposition so that the liver looks normal or healthy, at least in the absence or substantial absence of fat in hepatocytes. It is effective in delivering a valid amount of GS. The concentration and amount of GS depends on several factors, including substrate level in the subject and mode of administration, and can be determined empirically. Exemplary concentrations of GS in the compositions provided herein are, but are not limited to, (about) 0.1, 0.5, 0.6, 0.7, 0. .8, 0.9, 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000 or 5000 mg Concentrations of / mL GS or higher can be mentioned.

To formulate the GS composition, in one embodiment a weight fraction of GS is dissolved, suspended, dispersed or otherwise mixed in the selected medium at the desired concentration. The resulting mixture is a solution, suspension, emulsion and other such mixtures, including but not limited to solutions, suspensions, pastes, gels, aerosols, sprays, or systemic doses. It can be formulated as a non-aqueous or aqueous mixture containing any other formulation suitable for.

In general, GS compositions are prepared with regulatory approval in mind, or according to commonly recognized pharmacopoeia for use in animals and humans. The GS composition may include carriers such as diluents, excipients, or vehicles. Such pharmaceutical carriers may be sterile liquids such as water and oil. Aqueous saline and aqueous dextrose and glycerol solutions can also be used as liquid carriers, especially for injectable solutions. The composition, along with the active ingredient, is a diluent such as lactose, sucrose, dicalcium phosphate, or carboxymethyl cellulose; lubricants such as magnesium stearate, calcium stearate and talc; and binders such as starch. , Natural rubbers such as acacia rubber, gelatin, glucose, lactose, polyvinylpyrrolidin, cellulose and derivatives thereof, povidone, crospovidone, and other such binders known to those of skill in the art. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, choke, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene. , Glycerol, water, and ethanol. The GS composition is also preferably a small amount of wetting or emulsifying agent, or pH buffering agent, such as acetate, sodium citrate, cyclodextrin derivative, sorbitan monolaurate, triethanolamine sodium acetate. , Triethanolamine oleate, and other such agents. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.

Liposomal suspensions containing tissue-targeted liposomes may also be suitable as pharmaceutically acceptable carriers. These can be prepared by a method known to those skilled in the art. Liposomal delivery may also include sustained release formulations containing a pharmaceutical matrix such as collagen gel and fibronectin modified liposomes.

In addition to pharmaceutical compositions, GS can also be formulated or administered by other means, such as nutritional compositions such as dietary supplements, such as parenteral nutritional compositions (eg, alone or with other supplement ingredients). along with). Suitably, nutritional supplements may be provided in a dosing regimen such that sufficient GS reaches the intestine to provide a detoxifying effect on ammonia. GS may be included in such foods as a polypeptide (eg, a purified enzyme) or as part of an expression host cell or organism. Thus, for example, a microorganism (eg, yeast or bacterium or fungus) host cell or plant (including plant cells) may be engineered to express GS and itself, eg, whole cell or extract or other treatment. It may be administered as a substance (which can retain enzyme activity) or may be incorporated into a nutritional composition. Thus, for example, a bacterium or yeast cell suitable for human or non-human animal consumption may be engineered to express GS (ie, by introduction of a nucleic acid molecule containing a nucleotide sequence encoding GS). Alternatively, the plant may be manipulated in a similar manner and suitable plant portions and the like (eg, seeds, leaves, tubers, etc.) may be provided for administration. Which microorganisms (eg yeasts, bacteria, algae or fungi) are suitable for consumption by humans or other animals is known in the art and many such organisms are used today, for example in live fungal formulations. ing. Any such viable organism or formulation can be used on the basis of lactic acid bacteria such as, for example, Bifidobacterium or Lactobacillus species (eg Lactobacillus acidophilus). Thus, according to the invention, such organisms or preparations may be formulated for the GI tube and may be administered directly to the GI tube, for example by injection or infusion, or enema or rectal administration. The exact amount or dose of GS administered to a subject will depend on the activity of the GS, the route of administration, the disease or condition being treated, the number of doses, and other considerations such as the subject's weight, age and general condition. Specific dosages and dosing protocols can be determined empirically or, for example, estimated from tests in animal models. Exemplary therapeutically effective doses of GS include, but are not limited to, (about) 1 mg / kg to (about) 1000 mg / kg body weight per day, or (about) 10 mg / kg per day. Examples include (about) 0.1 mg / kg body weight per day to (about) 10,000 mg / kg body weight per day, including (about) 100 mg / kg body weight. Thus, for example, the subject is 0.1, 0.2, 0.3, 0.4, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70 per kg body weight per day. , 80, 90, 100, 200, 400, 600, 800, 1000, 2000, 4000, 6000, 8000, 10000, or 20000 mg or more of GS may be administered.

It is a feature of the present invention that GS is administered systemically. Therefore, GS can be administered by any method and route of systemic delivery of GS. In certain embodiments, the GS may be administered parenterally. Those skilled in the art include, but are not limited to, intravenous, intramuscular, intradermal, transdermal, subcutaneous, or intraperitoneal administration, devised in a manner suitable for each route of administration, and they. Appropriate methods of administration or delivery by any combination of any two or more of the above will be readily understood and selected. In some examples, the GS, or GS composition or product described herein, may be administered subcutaneously. In another example, the GS, or GS composition or product described herein, may be administered intravenously. For example, the GS composition can be administered intravenously by injection or infusion, such as an intravenous bolus.

Systemic administration may be oral. Oral administration means by oral delivery. Therefore, parenteral means that GS is not administered by ingestion through the mouth. Other means of administration that deliver the GS protein to the intestine, or more generally to the gastrointestinal (GI) tract, may be mentioned (eg, by direct administration to the rectum or enema, or GI tract). In a further embodiment, the invention does not include administration to muscles, especially skeletal muscle. Therefore, in such embodiments, the administration is not targeted at the muscle, i.e., non-muscle directed therapy.

GS can also be administered in combination or in combination with other therapeutic or active agents, in particular a second or additional therapeutic agent capable of treating (eg, ameliorating) fatty liver disease. The second or additional agent is typically an ammonia-lowering agent such as a nitrogen scavenger (or ammonia scavenger) or a substituted amino acid or urea cycle intermediate, or an analog thereof. Such agents may therefore contain amino acids such as arginine, glutamic acid, citrulline and / or ornithine, and / or N-acetylglutamic acid and / or the analog molecule carbamilglutamic acid [Carbaglu®]. More preferably, the second or additional agent is an ammonia lowering agent, more preferably a nitrogen scavenger. Such embodiments provide certain aspects of the invention.

The term "ammonia lowering agent" refers to a compound that removes ammonia, for example, from the blood of the subject being treated and / or reduces or inhibits ammonia production. Ammonia lowering agents can also be referred to as ammonia scavengers. The ammonia lowering agent may be an agent that reduces the blood concentration of ammonia. The ammonia lowering agent may be an agent or compound that increases the emission of ammonia or reduces its production in the subject. Ammonia emissions can be increased by binding to ammonia and providing compounds that are excreted. The ammonia lowering agent may be a small molecule. Ammonia lowering agents include nitrogen scavengers, ion exchange resins (eg Rylypsa), ammonia absorbers (eg Versantis, such as liposome-based ammonia absorbers), engineered microbiomes for removing ammonia (eg Synlogic), refaxmin. And may be selected from the group consisting of lactulose.

As used herein, the term "nitrogen scavenger" refers to a compound or agent that reduces the levels of nitrogen and / or ammonia in a subject by removing ammonia. Suitably, nitrogen scavengers reduce blood levels of ammonia. The terms ammonia scavenger and nitrogen scavenger are understood by those of skill in the art. In a suitable embodiment, the nitrogen scavenger can be metabolized to phenylacetylglutamine and then excreted in the urine to reduce the amount of nitrogen and / or ammonia in the subject (appropriately blood concentration).

In a suitable embodiment, the nitrogen trapping agent is a pharmaceutically acceptable salt of phenylacetic acid (also referred to herein as a phenylacetate salt), a pharmaceutically acceptable salt of phenylbutyric acid (also referred to herein as phenylbutyric acid). It may be selected from the group consisting of glycerol phenylbutyric acid (also referred to as a salt), a pharmaceutically acceptable salt of benzoic acid, a pharmaceutically acceptable prodrug thereof, and an ammonia binding resin. Other nitrogen scavengers will be well known to those of skill in the art.

As used herein, the term "pharmaceutically acceptable salt" refers to, for example, an acid addition salt of phenylacetic acid that is sufficiently basic, such as an inorganic or organic acid, such as hydrochloric acid, hydrogen bromide. Includes, for example, acid addition salts with acids, sulfuric acid, phosphoric acid, trifluoroacetic acid, formic acid, citric methane sulphonate or maleic acid. In addition, suitable pharmaceutically acceptable salts of sufficiently acidic phenylacetic acid are alkali metal salts such as sodium or potassium salts, alkaline earth metal salts such as calcium or magnesium salts, ammonium salts or pharmaceuticals. A salt with an organic base that provides an acceptable cation, such as a salt with methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris- (2-hydroxyethyl) amine.

In a suitable embodiment, the pharmaceutically acceptable salt of phenylacetic acid may be selected from the group consisting of sodium phenylacetate, potassium phenylacetate, ornithine phenylacetate.

In a suitable embodiment, the pharmaceutically acceptable salt of phenylbutyric acid may be selected from the group consisting of sodium phenylbutyrate and potassium phenylacetate.

In a suitable embodiment, the pharmaceutically acceptable salt of benzoic acid may be selected from the group consisting of sodium benzoate and potassium benzoate.

It is understood that the ammonia-lowering agent may be present in solvated and non-solvated forms, for example in hydrated form. It should be understood that all such solvated and non-solvated forms are included in the context of the present invention.

The term "prodrug", as used herein, is less active than an ammonia-lowering agent (such as a nitrogen scavenger) by chemical or biological moieties, but is metabolized to an ammonia-lowering agent. , Or an agent that is hydrolyzed in vivo to form an ammonia-lowering agent.

In particular, agents such as phenylacetic acid or phenylbutyric acid compounds that act to remove glutamine from the circulation (glutamine formed by the action of GS) are preferred. A second or additional agent, such as an ammonia lowering agent, may be administered individually, continuously or simultaneously with the GS protein, including in the same formulation or composition, or in separate compositions or formulations.

The second or additional agent may be administered by the same or different routes of administration, including oral. Thus, in one exemplary embodiment, a second or additional agent, such as an ammonia-lowering agent, may be administered orally or by other systemic means, and the GS may be administered by parenteral systemic means. It may be administered.

Ammonia lowering agents such as nitrogen trapping agents (including sodium phenylacetate, ornithine phenylacetate, sodium phenylbutyrate, or sodium benzoate) are used, for example, by intravenous infusion for acute management and / or for long-term maintenance, for example. May be orally administered to. i. v. The infusion may be peripheral, but central i. v. Injection is preferred. Similarly, amino acids such as arginine can be used orally or i. v. For example, the center i. v. It may be administered by infusion.

Such kits may be provided for use in the treatment of fatty liver disease. The components of the kit may be provided as a separate pharmaceutical composition comprising the agent in combination with one or more pharmaceutically acceptable carriers or excipients. The compositions of the present invention can be administered once or more than once. If administered more than once, the composition can be administered at regular intervals or as needed, eg as determined by the clinician. Constant intervals may include, for example, almost daily, weekly, biweekly, monthly, or any other interval. The choice of treatment protocol is well within the skill level of one of ordinary skill in the art. For example, the protocol can be determined based on tests on animal models. In another example, repeated doses of the composition may be administered to the subject if the ammonia level in the blood is above a given level.

The use of the GS protein in combination with the GS protein, or the use of an ammonia-lowering agent, according to the present invention is advantageous for the treatment of subjects for whom gene therapy is not appropriate or appropriate, such as children, or subjects who are refractory to gene therapy. Targets of such refractory may include, for example, those who have been previously exposed to, or have shown an immune response to, a viral vector used in the delivery of gene therapy.

Advantageously, the use of the protein as a therapeutic agent delivers higher doses of active protein to the subject, allowing the dose to be adjusted according to the subject and needs. Moreover, compared to gene therapy, protein therapy allows for a much faster response and is therefore more suitable for emergency use.

As already mentioned, one aspect of the invention relates to a pharmaceutical composition comprising an ammonia lowering agent for use in combination with a GS protein for use in the treatment of fatty liver disease. The ammonia-lowering agent can be formulated as a pharmaceutical composition for administration to a subject. Ammonia lowering agents are formulated in any conventional manner by mixing selected amounts of nitrogen scavengers with one or more physiologically or pharmaceutically acceptable carriers or excipients. Can be done. Appropriately, the composition may be for parenteral systemic administration or for oral administration. It will be appreciated that the pharmaceutical composition can also include a GS protein. In such an embodiment, the composition will be formulated for parenteral systemic administration.

The choice of carrier or excipient is within the skill of the dosing specialist and may depend on several parameters such as mode of dosing. In some examples, the ammonia lowering agent is provided as a liquid. In another example, the ammonia lowering agent is provided in dry form. Such dry forms can be rehydrated prior to administration by the addition of a suitable solution such as water, buffer, saline or other suitable solution. The ammonia-lowering agent may be formulated for direct administration, or for dilution or other modification. Therefore, the ammonia-lowering agent can be formulated in a single (or unit) or multiple dose form. Examples of single dose forms include ampoules and syringes. Examples of multiple dose forms include vials and bottles containing multiple unit doses.

The concentration of the ammonia-lowering agent in the formulation is effective in removing nitrogen and / or ammonium from the circulation when administered, or delivering an amount of ammonia-lowering agent effective in reducing or inhibiting ammonia production. Concentrations and amounts depend on several factors, including substrate levels in the subject and mode of administration, and can be determined empirically. Exemplary concentrations of the ammonia-lowering agent in the compositions provided herein are not limited to, but are (about) 0.1, 0.5, 0.6, 0.7. , 0.8, 0.9, 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000 Alternatively, a concentration of 5000 mg / mL ammonia lowering agent or higher can be mentioned.

To formulate the ammonia-lowering composition, in one embodiment a weight fraction of the ammonia-lowering agent is dissolved, suspended, dispersed or otherwise mixed in the selected medium at the desired concentration. The resulting mixture is a solution, suspension, emulsion and other such mixtures, including but not limited to solutions, suspensions, pastes, gels, aerosols, sprays, or systemic doses. It can be formulated as a non-aqueous or aqueous mixture containing any other formulation suitable for.

In general, ammonia lowering agents are prepared with regulatory approval in mind, or else according to generally recognized pharmacopoeia for use in animals and humans. The composition may include carriers such as diluents, excipients, or vehicles. Such pharmaceutical carriers may be sterile liquids such as water and oil. Aqueous saline and aqueous dextrose and glycerol solutions can also be used as liquid carriers, especially for injectable solutions. The composition, along with the active ingredient, is a diluent such as lactose, sucrose, dicalcium phosphate, or carboxymethyl cellulose; lubricants such as magnesium stearate, calcium stearate and talc; and binders such as starch. , Natural rubbers such as acacia rubber, gelatin, glucose, lactose, polyvinylpyrrolidin, cellulose and derivatives thereof, povidone, crospovidone, and other such binders known to those of skill in the art. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, choke, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene. , Glycerol, water, and ethanol. Phenylacetic acid, or a pharmaceutically acceptable salt composition thereof, may also be a small amount of wetting or emulsifying agent, or pH buffer, if desired, such as acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate. , Triethanolamine sodium acetate, triethanolamine oleate, and other such agents can also be included. Other examples of suitable pharmaceutical carriers will be known to those of skill in the art.

Ammonia lowering agents can be administered by any method and route of delivery of the compound to the body. In certain embodiments, the ammonia-lowering agent can be administered parenterally. Those skilled in the art include, but are not limited to, intravenous, intramuscular, intradermal, transdermal, subcutaneous, or intraperitoneal administration, formulated in a manner suitable for each route of administration, and they. Appropriate methods of administration or delivery by any combination of any two or more of the above will be readily understood and selected.

In addition to the pharmaceutical composition, the ammonia-lowering agent can also be formulated or administered by other means, eg, a nutritional composition such as a dietary supplement, eg, a parenteral nutritional composition (eg, alone or otherwise). With supplement ingredients). Appropriately, such compositions may be for oral or parenteral administration.

As already mentioned, the ammonia lowering agent is intended to be administered in combination with GS. It will be appreciated that the ammonia-lowering agent may be administered individually, continuously or simultaneously with the GS, including in the same formulation or composition, or in separate compositions or formulations. Thus, in one exemplary embodiment, the ammonia-lowering agent may be administered orally or by other systemic means, and the GS may be administered by parenteral systemic means.

Exemplary therapeutically effective doses of ammonia lowering agents are, but are not limited to, (about) 10 mg / kg to about 1000 mg / kg body weight per day, or about 100 mg / kg to about 500 mg per day. Included is about 1 mg / kg body weight per day to (about) 2000 mg / kg body weight per day, including / kg body weight. Therefore, for example, the subject is 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300 per kg of body weight per day. , 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or 2000 mg or more of the ammonia lowering agent may be administered. Other exemplary therapeutically effective doses of ammonia lowering agents include, but are not limited to, from about 1 g / day to about 50 g / day. Thus, for example, a subject may be administered 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50 grams or more of an ammonia-lowering agent per day. It will be appreciated that the effective dose may vary depending on the ammonia lowering agent. The GS composition and / or the composition comprising an ammonia-lowering agent or a composition comprising an additional agent that is not an ammonia-lowering agent can contain one or more unit dosage forms, if desired, with a needle. It may be presented in a package, kit or dispenser device, such as a syringe, or a vial and a syringe with a needle. The kit or dispenser device may be accompanied by an administration manual. In embodiments where the GS composition and the composition comprising the ammonia lowering agent are separate, the kit may comprise a composition comprising the GS composition and the ammonia lowering agent. Suitably, in such an embodiment, the kit may include a GS composition and a nitrogen scavenger. Suitably, in such an embodiment, the kit may include a GS composition and a composition comprising a phenylacetic acid salt, such as sodium phenylacetate. The composition is a packaging material, a composition, and a product containing a label indicating that the composition is intended to be administered to a subject to treat fatty liver disease or a disease or condition associated with fatty liver disease. Can be packaged as.

Suitable systemic administration methods will be known to those of skill in the art. As an example, systemic administration may be achieved by parenteral routes of administration, such as intravenous or subcutaneous routes. It will be appreciated that "systemic administration" allows expression vector products (such as glutamine synthetase or biologically active fragments thereof) to be expressed within multiple sites of a patient. In the context of the present invention, systemic administration does not include intramuscular administration.

GS for use in the treatment of fatty liver disease (unless otherwise required by the context), GS protein for use in combination with ammonia lowering agents, for use in combination with GS It will be appreciated that the embodiments described with respect to ammonia lowering agents, their use, therapeutic methods, compositions, kits and products are generally applicable to the remaining aspects of the invention.

The invention is further described with reference to the following non-limiting examples and figures.

FIG. 1 shows the results of size exclusion chromatography (SEC) on a Superose 12 column of a 20 kDa N-terminal aldehyde PEG conjugate of a human GS protein prepared in Example 1. The graph shows that the multimer was eluted in fractions 8 and 9, and the monomer was eluted in fraction 10. FIG. 2 shows a comparison of in-vitro GS activity of various GS candidates: non-conjugated GS (wt GS) and PEG-conjugated variants (GS1, GS2, GS3, GS4) contrasted with negative controls. .. Glutamine synthetase activity is shown as OD570 nm according to the assay of Acosta et al., 2009, World J. Gastroenterol., 15 (23), 2893-2899. GS1- (N-terminal aldehyde monomer); GS2-Nof-20 ™; GS3-Nof-30 ™, GS4-N-terminal aldehyde monomer. FIG. 3 shows plasma before and after administration of male, wild-type (wt) CD1 mice of various conjugates at (A) baseline, (B) 24 hours post-administration, and (C) 72 hours post-administration. The PEG ELISA results for are shown. (1-N-terminal aldehyde-conjugated GS monomer; 2-Nof-20 ™ GS-conjugated multimer; 3-Nof-30 ™ conjugated GS multimer; 4--N-terminal aldehyde-conjugated PEG mass body). FIG. 4 shows the results of GS activity (OD535 nm) in liver lysates in wt CD1 mice administered at 2.5 mg / kg, 3 days after administration, as described in Example 3. FIG. 5A shows the results of the liver GS activity assay. FIG. 5B shows the plasma GS activity assay results. Both liver and plasma GS activity were assayed in GS protein, and bile duct ligation (BDL) rats treated with GS protein and nitrogen scavengers. FIG. 6 shows stained liver biopsies (with hematoxylin and eosin) of treated and untreated OTC mice. Recombinant human GS protein-administered mice exhibit normal liver structure compared to non-administered mice. FIG. 7 shows the ammonia levels of the control and GS-treated mice described in Example 6. FIG. 8 shows the fat percentage of liver sections in controls by digital FPA analysis in GS (AM 535) -treated MCD mice described in Example 6. FIG. 9 shows the results of the graph-type HFHC rat CPA (collagen proportional area) according to Example 7. FIG. 10 shows the effect of Example 7 on fibrosis after 7 weeks of double weekly administration of GS enzyme (AM-535) with Sirius red staining; same CPA levels as normal non-HFHC diets. Shows.

Example 1
Preparation and Purification of GS Protein and GS Protein-PEG Conjugate Human Glutamine Synthetase (GS) Preparation: Contains the 5'sequence that encodes the human GS gene (His tag and linker GGGGS at the N-terminus of GS) and is expressed in bacteria. ^{A pET30a +} vector containing the codon-optimized SEQ ID NO: 5) was used in the Escherichia coli expression system. After plasmid construction, evaluation of GS expression was performed at a wide range of induction (IPTG) and expression temperatures. Human GS was solublely expressed in the construct detected by SDS-PAGE. Soluble protein was extracted from cells using lysis buffer (50 mM Tris pH 8.0, 10% glycerol, 0.1% Triton X-100, 100 ug / ml lysozyme, 1 mM PMSF, 3 units of DNAse, 2 mM MgCl). .. Soluble protein was extracted after centrifugation. After the expression test, the best conditions were found in BL21 (DE3) cells, cultured and induced at 25 ° C. for 16 hours with 0.1 mM IPTG. Other conditions tested included the use of various IPTG leads (0.01M to 0.1M IPTG), various incubation temperatures (ranging from 16 ° C to 37 ° C), and induction incubation times of 4 to 16 hours. rice field.

Purification of expressed GS: The first purification step of the expressed protein included His tag purification with Ni-NTA beads, washing with 20 mM imidazole, and elution with 300 mM imidazole.

Protein PEG fusion: GS proteins were conjugated to N-terminal aldehyde 20 kDa peg under reducing conditions (using 20 mM sodium cyanoborohydride) for 16 hours (Dr Reddy's 20 kDa N-terminal Aldehide PEG).

Final purification: The conjugated protein was further purified using SEC chromatography. A Superior 6 or Superior 12 column (see FIG. 1) was used. Multimers were found at fractions 8 ± 9. Fraction 10 of Superiore 12 contained a (low concentration) multimer. In Superiore 6, the multimer was found in the fraction 8 + 9 and the monomer was found in the fraction 12/13.

A final formulation of PBS containing trehalose and sucrose, GS in pH 7.4 was prepared.

Example 2
Activity of GS preparation
Examples using the assay of Acosta et al., 2009 (see above) modified from the original assay described in Ehrenfeld et al., 1963, J. Biol. Chem. 238 (11), 3711-3716. Various GS preparations and PEG conjugates prepared according to 1 were tested for GS activity.

100 ug of purified protein sample was added to the following reaction buffers: 150 μL stock solution (100 mmol / L imidazole-HCl buffer [pH 7.1], 40 mmol / L MgCl2, 50 mmol / L, β-mercaptoethanol, 20 mmol /). LATP, 100 mmol / L, glutamate, and 200 mmol / L hydroxylamine adjusted to pH 7.2). The tubes were incubated at 37 ° C for 15 minutes. The reaction was stopped by adding 0.6 mL [2 × concentration] ferric chloride reagent (0.37 mol / L FeCl3, 0.67 mol / L HCl and 0.20 mol / L trichloroacetic acid). The sample was placed on ice for 5 minutes. The precipitated protein was removed by centrifugation at 10,000 g and the absorbance of the supernatant was read at 535-570 nm with respect to the reagent blank. The results are shown in FIG. Trin1- (20 kD size N-terminal aldehyde monomer PEG obtained from Dr Reddy's); monofunctional linear 20 kD PEG, NHS active ester and GS protein conjugated to Trin2-NOF corporation. Nof-20 conjugated GS); Monofunctional linear 30 kD PEG, NHS active ester and GS protein conjugated Nof-30); Trin4-N-terminal aldehyde, GS multimer obtained from Trin3-NOF corporation. ). The activities of the other conjugates were similar, but Trin4 (N-terminal aldehyde multimer) showed the best activity and the activity profile was very similar compared to wt GS (non-conjugated).

Example 3
Administration of GS Protein to Mice-Effects of GS Protein-PEG Conjugate on Plasma Levels Various GS protein and PEG conjugates prepared as described in Example 1 in male wild-type (wt) CD1 mice. Subcutaneous (Trin1-N-terminal aldehyde-conjugated GS monomer; Trin2-Nof-20 GS-conjugated multimer; Trin3-Nof-30-conjugated GS multimer; Trin4-N-terminal aldehyde-conjugated PEG multimer) s.c) It was administered at 2.5 mg / kg by administration. ELISA was performed according to the protocol outlined by the manufacturer (Abcam PEG ELISA kit, ab133605). The plasma ELISA results shown in FIG. 3, as expected, show very low or undetectable levels at all time points for non-conjugated wt GS. After 24 hours, some candidates were found to have high levels in plasma, but 72 hours after administration, Trin-GS 4 (N-terminal aldehyde-conjugated PEG GS multimer) was the highest. showed that. N = 2 animals in each group. Therefore, this experiment has shown that systemic administration of GS protein can be successfully used to obtain high circulating levels of GS PEG conjugates, and especially at levels that can be therapeutically effective or active.

Example 4
Administration of GS protein to mice-GS activity level of liver lysates The activity assay was performed except that 500 μg of liver lysates (derived from the slaughtered mice from the experiment of Example 3) were added to each reaction as needed. Was performed as described in Example 2. The results are shown in FIG. GS activity results in liver lysates 3 days after administration showed that the excellent candidate was the N-terminal aldehyde-conjugated PEG GS multimer, showing significant activity above baseline compared to vehicle (saline) controls. It shows that it was the only candidate. N = 2 animals in each group.

Example 5
^{An Otc spf-ash} mouse model of urea cycle disorders (OTC deficiency) was used to show the effects of GS and GS + SP. For details on the mouse used, see https: // www. jax. It can be found in org / straight / 001811 (B6EiC3Sna / A-Otc ^{spf-ash / J).} Feed the mice a normal diet. The ages varied from about 10 to 23 weeks, and the groups were well matched. All animals are male hemizygous (OTC is X-linked and therefore only presents on the male X chromosome, hence mice are knocked out).

A non-pegliated human GS containing an N-terminal GGGS linker and a GGGGS linker was used. This is referred to in FIG. 6 as AM535. All groups [medium, GS and GS + SP; GS = glutamine synthetase, GS + SP = glutamine synthetase + sodium phenylacetate] were treated as follows:
The experiment was conducted from Tuesday to the following Wednesday (8th).
SP is 350 mg / kg twice daily, i. p. Administered; GS was administered to all treatment groups for the first 4 days (once daily at 40 mg / kg ip), followed by a 2-day [weekend] discontinuation, 40 mg / kg i. p. GS was administered for another 3 days.

Mice were slaughtered on day 8, blood was extracted, plasma was deposited, and this plasma was used for ammonia quantification (see method below).

Genotyping is performed using standard methods described in the literature.

Materials and Methods All experiments were performed in accordance with the 1986 Animal (Scientific Treatment) Act revised according to European Directive 2010/63 / EU. All animals are humane according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals (National Institute of Health Sciences Publications 86-23; 1985). Received care. All animals used in these experiments were male Sprague-Dawley rats (body weight at the start of the experiment, 250 g) obtained from Charles River Laboratories (Kent, UK) and were divided into 5 groups: biliary ligated animals + ammonia. + Physiological saline serum (BDL + HA + SS, n = 6), bile duct ligated animal + ammonia + sodium phenylacetate (BDL + HA + SP, n = 6), bile duct ligated animal + ammonia + sodium phenylacetate + glutamine synthetase (BDL + HA + SP + GS, n = 5) , Bile duct ligated animal + ammonia + glutamine synthetase (BDL + HA + GS, n = 6), animal undergoing sham surgery + glutamine synthetase (SHAM + GS, n = 5). Treatments that include SP and GS may be referred to as "COMBO".

Bile duct ligation surgery Under general anesthesia (100% oxygen 5% isoflurane for induction, 2% isoflurane in air for maintenance), rats undergo triple ligation of the bile duct to induce chronic liver injury (small laparotomy method) and surgery. Tested after 28 days. Midline laparotomy was performed under anesthesia. In the BDL group, the common bile duct was isolated, triple-ligated with 3-0 silk thread, and incised between the ligated parts. The sham surgery group performed the same procedure without an incision between the ligatures. After BDL, all animals continued to gain weight, comparable to sham surgery controls. The overall mortality rate in both groups was less than 10% and occurred within 36 hours of surgery.

Non-cirrhotic hyperammonemia conditions Twenty-three rats were treated with a hyperammonemia (HA) diet. The amino acid recipes used for about 100 g of stock are 15 g leucine, 7.7 g phenylalanine, 7 g glutamic acid, 10 g alanine, 4.4 g proline, 5.8 g threonine, 11 g aspartic acid, 5 g serine, 4.8 g glycine, 3.3 g. It was arginine, 9.6 g lysine, 8.4 g histidine, 3 g tyrosine, 1.5 g tryptophan, and 10.6 g valine. 25 g of this mixture (1: 5 mixed with standard rodent dietary powder) was freshly prepared daily and rats had free access to it for 5 days. The recipe was similar to the amino acid composition of rodent hemoglobin, which mimics the effects of [2], [1] gastrointestinal bleeding, which are known to result in systemic hyperammonemia.

Sodium phenylacetate condition Eleven rats were given a sodium phenylacetate (SP) diet. 0.3 g / kg daily was mixed with dietary powder for 5 days and freshly prepared daily.

Glutamine Synthetase Conditions 16 rats were injected intraperitoneally with GS once every two days (day 1 and day 3). The total volume injected is 3 ml i. Which allows GS of 18-22 mg / kg. p. Met.

Results Administration of GS Protein to Mice-GS activity levels in liver and blood Activity assays were performed as described in the Materials and Methods section above. The results are shown in FIGS. 5A and 5B. The results of rat liver measured on the 5th day show that the GS activity is the best in the SHAM + GS group. Furthermore, it can be seen from FIG. 5A that GS and GS + SP treatment increases GS activity in the liver of mice that have undergone BDL. When measured with blood, the results show that the BDL + GS group has the best GS activity. Furthermore, it can be seen from FIG. 5B that the GS activity in the blood is consistent over time, even 24 and 48 hours after administration.

Hematoxylin and Eosin (H & E) Staining of OTC Mouse Liver The staining method involves the application of hemaram, a complex formed from aluminum ions and oxidized hematoxylin. This stains the nucleus of the cell blue. Nuclear staining is followed by counterstaining with an aqueous or alcoholic solution of eosin Y. Counterstain dyes other acidophilic structures in various colors of red, pink and orange. Nucleus staining with hemalam does not require the presence of DNA and is probably due to the binding of dye-metal complexes to arginine-rich basic nucleoproteins such as histones. Eosin is a fluorescent red dye resulting from the action of bromine on fluorescein. Eosin can be used to stain the cytoplasm for microscopic experiments. Structures that are easily dyed with eosin are called acidophilic. Eosin is most often used as a counterstain with hematoxylin in H & E (hematoxylin and eosin) staining. Eosin strongly stains red blood cells red. Eosin is an acidic pigment that appears in the basic part of the cell, the cytoplasm.

Liver specimens stained with H & E therefore typically show blue nuclei, and the cytoplasm and extracellular matrix are different colors pink. After H & E staining, the present inventors took photographs using a 40 × optical microscope and appropriately compared the treated group and the non-administered group. In this protocol, tissues were taken from animal experiments, stored in paraffin, sliced with a microtome (about 4-9 μm sections), placed on slides, and prepared for staining and / or IHC analysis.
Solution / Buffer> 98% Xylene (Fiser Extra Pure SLR Fiser Chemical X / 0200/17)
> 98% propanol (Sigma Aldrich, Catalog No. I9516)
H2O (distillation)
Hematoxylin solution (Sigma Aldrich, catalog number MHS1)
Eosin Y solution (Sigma Aldrich, catalog number E4009)

For this experiment, mice (OTC semi-zygous males) were intraperitoneally administered at 40 mg / kg of recombinant human GS protein at 40 mg / kg for 8 days (previously described), and after culling, the liver was recovered. Objectives of this test: 1. Recombinant human GS (AM-535), 2. Mouse livers were divided into two groups for control / medium. Both groups of mouse livers were examined for underlying pathology, including H & E staining.

Fatty liver disease / fatty liver-like disease has been observed in all 6 enzyme-related urea cycle disorders [Bigot et al. 2017'Liver involvement in urea cycle disorders: a review of the literature]. The urea cycle has been shown to be dysregulated in NAFLD (Non-alcoholic fatty liver disease) [Chiara et al 2018'Urea cycle dysregulation in nonalcoholic fatty liver disease].

The paraffin texture block (immobilization and paraffinization) was cooled at −10 ° C. on a Lamb cooling table (model TC-10). The sample was sliced with a microtome. Liver sections were 4 μm thick; kidneys were 4 μm; brain tissue was sliced at 8 μm. After slicing, the tissue was placed in a heated water tank (heated to 55 degrees Celsius) for a few seconds and then placed on a slide (the tissue was soaked in water with the slide and placed on the slide).
1. 1. Deparaffinized tissue: The tissue is "dunked" in xylene for 45 seconds.
2. 2. Remove and fix xylene: "immerse" in propanol for 45 seconds
3. 3. Hematoxylin staining: Drop 1 drop of hematoxylin staining per 50-100 mm2 and wait for 45 seconds (usually about 5 drops are required per slide).
4. Washing with hematoxylin: Rinse the hematoxylin residue strongly with warm (30 ° C) water (distilled H2O recommended) for about 45 seconds. Eosin staining: Drop 2-3 drops of eosin staining and wait for 30 seconds. Eosin wash: Rinse again with cold water (distilled H2O) for 15 seconds 7. Final wash: Soak in propanol for 45 seconds, then in xylene for 45 seconds

The result should be blue for the nucleus and pink to red for the cytoplasm.

The recombinant human GS-treated mice shown (see Figure 6) showed normal liver structure and cellular integrity, had little fibrosis, and had a fairly healthy liver overall. In contrast, mice in the control (non-administered) group showed significantly less healthy liver suggestive of steatosis and fatty liver-like disease.

Hepatocyte steatosis is either the presence of a single large lipid droplet coexisting with nuclear translocation to the periphery of the cell (large droplet lipid disease), or small lipid droplets and no nuclear displacement (small droplet lipopathy). Can be identified by. The former is typical of NAFLD and the latter is associated with NASH inflammation. These results demonstrate that both NAFLD and NASH can be involved in this OTC mouse model, as well as a clear and extremely significant improvement in liver health of treated animals (see Figure 6). ).

These results indicate that GS therapy is a potential tool for treating and improving liver steatomatosis, including fatty liver, NASH (non-alcoholic steatohepatitis) and related liver disease disorders. There is. At short-term administration (8 days), the fibrosis-like phenotype of AM-535-administered (recombinant GS enzyme) (OTC semi-zygous) mice was significantly different from that of control mice, and fatty liver type progression occurred in this short period of time. It was shown that improvement and inhibition occurred. This is the first and most powerful sign that GS therapy can be useful in the treatment and improvement of steatoses and related disorders (including NAFLD, NASH and fibrosis).

Example 6
Mouse MCD Test The most widely used diet for inducing NAFLD / NASH is the methionine-choline deficient (MCD) diet. A standard MCD diet also has a high content of sucrose (40% of energy) and is reasonably rich in fat (10-20%). This is an extremely reproducible model [5].

A pegged human recombinant GS containing an N-terminal GGGS-HHHHH-GGGGS linker / tag sequence was used. Tests were conducted to determine whether this GS (referred to as AM535 in Figure 7) is effective in this model and has the potential to reduce fat and be a potential candidate for NAFLD / NASH application. did.

Method Mouse model background, phenotype:
Mouse: C57 / Black
Gender: Female Weight: 25g-30g
Age:> 7 weeks MCD diet dose:
5 g / day / mouse + drug intervention as needed MCD diet composition:
Methionine / choline deficient diet composition (amount g / kg diet):
Sucrose 455.3gm
Cornstarch 200.0gm
Corn oil 100.0gm
Alphacell Non-Nutritive Bulk 30.0gm
AIN 76 Mineral Mix 35.0gm
AIN 76 Mineral Mix Composition (gm / kg):
Dicalcium Phosphate 500.00 gm
Sodium chloride 74.00 gm
Tripotassium citrate monohydrate 220.00 gm
Potassium sulfate 52.00 gm
Magnesium oxide 24.00 gm
Manganese carbonate (43-48% Mn) 3.50 gm
Iron citrate oxide (16-17% Fe) 6.00 gm
Zinc oxide (70% ZnO) 1.60 gm
Copper carbonate (53-55% Cu) 0.30 gm
Potassium iodate 0.01 gm
Sodium selenite 0.01 gm
Potassium chromium sulfate 0.55 gm
Sucrose, fine powder 118.00 gm-second calcium phosphate 3.0 gm, L-alanine 3.5 gm, L-arginine hydrochloride 12.1 gm, L-asparagin monohydrate 6.0 gm, L-aspartic acid 3 .5 gm, L-cystine 3.5 gm, L-glutamate 40.0 gm, glycine 23.3 gm, L-histidine hydrochloride 4.5 gm, L-isoleucine 8.2 gm, L-leucine 11.1 gm, L-lysine hydrochloride 18.0 gm, L-phenylalanine 7.5 gm, L-proline 3.5 gm, L-serine 3.5 gm, L-threonine 8.2 gm, L-tryptophan 1.8 gm, L-tyrosine 5.0 gm, L-valine 8 .2 gm, DL-alpha-tocopherol acetate (250 u / gm) 0.484 gm, vitamin A palmitate (250,000 u / gm) 0.0792 gm, vitamin D3 (400,000 u / gm) 0.0055 gm, ethoxykin 0. 02gm, Vitamin Mix-Biotin 0.0004gm, D-Calcium Pantothenate 0.0661gm, Folic Acid 0.002gm, Inositol 0.1101gm, Menadion 0.0496gm, Niacin 0.0991gm, p-Aminobenzoic Acid 0.1101gm, Pyridoxin Hydrochloride Salt 0.0220 gm, Riboflavin 0.022 gm, Thiamine hydrochloride 0.022 gm, Vitamin B12 (0.1% trit.) 0.0297 gm, Ascorbic acid 1.0166 gm, Corn starch 3.4503 gm

Method / test overview:
MCD mice were kept in a comfortable condition and housed in a feeding facility for at least one week before starting feeding. The test lasted 2.5-4 weeks, depending on individual requirements.

Therapeutic intervention was performed according to the instructions for use. Blood ammonia was collected together with the tissue at the time of culling and histological examination was performed. This MCD test was performed for 18 days and GS twice a week as defined above i. p. Was given at 25 mg / kg. In total, mice received 6 doses of GS as defined above. Organs and blood were collected for analysis.

Test group:
-2 x Medium control (injection of saline)
-3 x GS administration

Post-slaughter analysis:
Using 10 ul of mouse blood (fresh)-Ammonia was measured by the Ammonia Fuji checker according to the manufacturer's instructions. Briefly, 10 ul was applied to the cartridge and inserted into a Fuji machine. The machine (containing bromophenol blue) heats the entire blood sample. When the sample is heated, NH3 gas is released from whole blood and mixed with bromophenol blue. The sample is then read by a Fuji checker at OD 600 nM to obtain an estimate of ammonia (micromol / uM), which is shown to the console operator 2 minutes after insertion of the cartridge.
[https://www.fujifilm.com/products/medical/fdc/pdf/index/NH3-W_9903230-A4.pdf].

Livers were analyzed by H & E staining described in Example 5 and fat counting was performed digitally according to the method outlined in [6,7] (mFPA).

Results Ammonia level vs. control mice Immediately after slaughter, ammonia levels were measured, 10 microliters of whole blood was collected from each mouse and analyzed with a Fuji ammonia checker. Ammonia was significantly elevated in MCD-borne control mice (considered pathogenic at about 100 uM), and GS-treated animals dropped to very low baseline (non-pathogenic) levels of ammonia (mean about 40 uM). It showed a very large drop in level (see Figure 7). This decrease was shown to be very significant.

H & E staining and fat counting:
H & E staining was performed according to the protocol of Example 5 described above. Liver sections were then digitally analyzed and fats were counted in the dose group vs. the control group.

It could be seen from the independent mFPA fat counts that the treated animals were significantly lower in fat when compared to the vehicle control animals (see Figure 8). Therefore, it can be concluded that GS significantly reduces liver fat in this MCD model.

Summary GS has been found to reduce ammonia levels very significantly (from pathogenic levels of ammonia (about 100 uM) to low non-pathogenic levels (about 40 uM)). This is the third model in which GS showed a very significant reduction in ammonia levels. Therefore, GS may be an excellent candidate for further pursuit in hyperammonemia-related conditions, including urea cycle disorders, hepatic encephalopathy and organic acidemia.

From H & E staining and subsequent fat counting, we also showed that recombinant GS significantly reduced fat levels. Both MCD and OTC mouse models showed reduced fat levels compared to vehicle controls.

Example 7
High-fat high-cholesterol rat principle / background High-fat high-cholesterol (HFHC) rats are models of progressive NAFLD (non-alcoholic fatty liver disease), steatohepatitis, fibrosis and final cirrhosis [9-11]. ..

Objective: The last 7 weeks of the 16-week model, twice a week, i. p. To determine if AM-535 administered (at about 10 mg / kg) alters the course of this model and improves disease progression and outcome.

Outline / Method of Test 8-week-old male Sprague Dawley rats were obtained from Charles River, UK. Rats were housed and kept in a comfortable condition for at least one week prior to the test. 220-250 g of male SD rats were fed a high-fat, high-cholesterol (100 g) or solid feed (100 g) diet for 16 weeks, and a pegged human recombinant GS containing an intraperitoneal GS (N-terminal GGGS-HHHHH-GGGGS linker / tag sequence). , AM-535) or media were randomly assigned to receive twice a week, including weeks 9-16. After slaughter, blood was obtained by heart puncture and the organs were collected for analysis and pathological examination.
group:
・ Normal solid feed food (+ medium) n = 4
・ HFHC diet (+ medium [saline]) n = 4
・ HFHC diet (+ GS) n = 4
Total number of animals in the treatment test n = 12

High-fat, high-cholesterol diet composition

Liver tissue was stored in paraffin, sliced with a microtome (about 8 μm section) and placed on a slide for staining. Sirius red staining was performed to detect collagen: This technique is based on a strong bond between the dye sulfonic acid group and the basic group of collagen fibers.
Briefly, the staining protocol is:
1. 1. Paraffin sections of the liver were dewaxed and hydrated; 2. 2. Staining with picrosirius red solution ("Direct 80", Catalog No. 2610-10-8) for 1 hour; The sections were shaken vigorously or soaked very gently with a damp filter paper; 4. 100% ethanol was replaced 3 times to dehydrate the sections. Finally, the sections were transparent with xylene, mounted with DPX encapsulant and imaged.

result

表１．血中アンモニアおよび質量。アンモニアはいずれの群でも上昇しないことに注意。肝臓：体重比平均も媒体群と比べて治療群で若干低かった（０．０４７ｖｓ０．０５１）が、これは統計的に有意ではなかった。

Table 1. Blood ammonia and mass. Note that ammonia does not rise in either group. The liver: body weight average was also slightly lower in the treatment group compared to the vehicle group (0.047 vs 0.051), but this was not statistically significant.

Blood ammonia results were not elevated with respect to ammonia in any group (including vehicle HFHC rats), with a slight decrease in liver: body weight ratio seen in GS (AM-535) treated group HFHC rats when compared to vehicle. Showed that it was done.

Liver pathology examination Collagen proportional area (CPA) results FIG. 9 shows that the CPA (collagen proportional area) was significantly reduced in GS 7 weeks after the administration twice a week, and the same CPA level as that of normal diet animals could be shown. It is shown that.

Analysis showed a significant reduction in CPA (collagen proportional area) in HFHC AM-535-treated animals (Sirius Red-stained liver sections). Collagen proportional area (CPA) measurement quantifies the fibrous tissue of a liver biopsy or section (human patient or animal) by measuring the amount of collagen deposits as a percentage of the total biopsy area [11,12]. CPA predicts clinical outcomes in patients with fatty liver and cirrhosis [13,14].

Fibrosis is the formation of an abnormally large amount of scar tissue in the liver. Fibrosis occurs when the liver attempts to repair and replace damaged cells. Therefore, fibrosis is a marker of late fatty liver disease and cirrhosis.

Summary In this particular study, HFHC rats were fed a high-fat, high-cholesterol diet for 16 weeks (or a normal diet for naive control animals). In the last 7 weeks, HFHC diet rats were dosed with either vehicle (saline) or GS twice weekly at about 10 mg / kg. After slaughter, clinical blood parameters were measured. When GS HFHC rats were compared to vehicle HFHC controls, similar levels were observed for most blood and mass parameters.

In contrast to vehicle HFHC animals where CPA was measured at very high levels, GS was able to significantly reduce CPA to normal diet levels in rats fed the HFHC diet. The HFHC model is not a hyperammonemia model because ammonia did not rise in either group. Moreover, given the extremely low toxicity and high safety profile of this enzyme therapy, it is feasible to increase the dose from 10 mg / kg twice weekly. GS administration was started at 7 weeks. These data are particularly interesting in light of the fact that at 9 weeks, rats would have already developed liver disease, indicating that GS is a potential treatment for liver fibrosis.

(Reference)
[1] Riggs A. The amino acid composition of some mammalian hemoglobins: mouse, guinea pig, and elephant. J Biol Chem 1963; 238: 2983-2987.

[2] Balata S, Olde Damink SW, Ferguson K, Marshall I, Hayes PC, Deutz NE, Williams R, Wardlaw J, Jalan R. Induced hyperammonemia alters neuropsychology, brain MR spectroscopy and magnetization transfer in cirrhosis. Hepatology 2003; 37: 931-939.

[3] Stewart-Wallace AM. A biochemical study of cerbral tissue, and of changes in cerebral oedema. Brain 1939; 62: 426-38.

[4] Traber PG, Ganger DR, Blei AT. Brain edema in rabbits with galactosamine-induced fulminant hepatitis. Regional differences and effects on intracranial pressure. Gastroenterology 1986; 91: 1347-56.

Kiernan JA (2008) Histological and Histochemical Methods: Theory and Practice. 4th ed. Bloxham, UK: Scion.
Lillie RD, Pizzolato P, Donaldson PT (1976) Nuclear stains with soluble metachrome mordant lake dyes. The effect of chemical endgroup blocking reactions and the artificial introduction of acid groups into tissues. Histochemistry 49: 23-35.
Llewellyn BD (2009) Nuclear staining with alum-hematoxylin. Biotech. Histochem. 84: 159-177.

[5] Machado et al; PLoS One. 2015; 10 (5): e0127991
[6,7] Hall et al, Liver Int. 2014, Oct 34 (9) 1417-1427 and Hall et al, Liver Int. 2013 Jul 33 (6) 926-935
[8] Marques et al Adipocyte 2016 5 (1) 11-21
[9] Ichimura et al Hepatology Research Vol 45 Issue 4 2015 458-469
[10] Jensen et al Diabetology and Metabolic Syndrome 10 4 (2018)
[11] Buzetti et al Alimentary Pharmacology and Therapeutics Vol 49 Issue 9 1214-1222
[12] Hall et al, Histopathology 2013 62 (3) 421-430
[13] Xie et al Heptobiliary Pancreat Dis Int 2011 10 (5) 497-501
[14] Huang et al Liver Int 2013 Sept 33 (8) 1249-1256
arrangement:
SEQ ID NO: 1 [Complete human protein]
MTTSASSHLNKGIKQVYMSLPQGEKVQAMYIWIDGTGEGLRCKTRTLDSEPKCVEELPEWNFDGSSTLQSEGSNSDMYLVPAAMFRDPFRKDPNKLVLCEVFKYNRRPAETNLRHTCKRIMDMVSNQHPWFGMEQEYTLMGTDGHPFGWPSNGFPGPQGPYYCGVGADRAYGRDIVEAHYRACLYAGVKIAGTNAEVMPAQWEFQIGPCEGISMGDHLWVARFILHRVCEDFGVIATFDPKPIPGNWNGAGCHTNFSTKAMREENGLKYIEEAIEKLSKRHQYHIRAYDPKGGLDNARRLTGFHETSNINDFSAGVANRSASIRIPRTVGQEKKGYFEDRRPSANCDPFSVTEALIRTCLLNETGDEPFQYKN
Sequence 2 (only methionine is cleaved due to mature protein in vivo):
TTSASSHLNKGIKQVYMSLPQGEKVQAMYIWIDGTGEGLRCKTRTLDSEPKCVEELPEWNFDGSSTLQSEGSNSDMYLVPAAMFRDPFRKDPNKLVLCEVFKYNRRPAETNLRHTCKRIMDMVSNQHPWFGMEQEYTLMGTDGHPFGWPSNGFPGPQGPYYCGVGADRAYGRDIVEAHYRACLYAGVKIAGTNAEVMPAQWEFQIGPCEGISMGDHLWVARFILHRVCEDFGVIATFDPKPIPGNWNGAGCHTNFSTKAMREENGLKYIEEAIEKLSKRHQYHIRAYDPKGGLDNARRLTGFHETSNINDFSAGVANRSASIRIPRTVGQEKKGYFEDRRPSANCDPFSVTEALIRTCLLNETGDEPFQYKN
SEQ ID NO: 3 cDNA
CGAGAGTGGGAGAAGCGGAGCGTGTGAGCAGTACTGCGGCCTCCTCTCCTCTCCTAAC
CTGCTCTCGCGGCCTACCTTTACCCGCCCGCCTGCTCGGCGACCAGAACACCTTCCACCA
TGACCACCTCAGCAAGTTCCCACTTAAATAAAGGCATCAAGCAGGTGTACATGTCCCTGC
CTCAGGGTGAGAAAGTCCAGGCCATGTATATCTGGATCGATGGTACTGGAGAAGGACTGC
GCTGCAAGACCCGGACCCTGGACAGTGAGCCCAAGTGTGTGGAAGTTGCCTGAGTGGA
ATTTCGATGGCTCCAGTACTTTACAGTCTGAGGGTTCCAACAGTGACATGTATCTCGTGC
CTGCTGCCATGTTTCGGGACCCCTTCCGTAAGGACCCTAACAAGCTGGTGTTATGTGAAG
TTTTCAAGTACAATCGAAGGCCTGCAGAGACCAATTTGAGGCACACCTGTAAACGGATAA
TGGACATGGTGAGCAACCAGCACCCCTGGTTTGGCATGGAGCAGGAGTATACCCTCATGG
GGACAGATGGGCACCCCTTTGGTTGGCCTTCCAACGGCTTCCCAGGGCCCCAGGGTCCAT
ATTACTGTGGTGTGGGAGCAGACAGAGCCTATGGCAGGGACATCGTGGAGGCCCATTACC
GGGCCTGCTTGTATGCTGGAGTCAAGATTGCGGGGACTAATGCCGAGGTCATGCCTGCCC
AGTGGGAATTTCAGATTGGACCTTGTGAAGGAATCAGCATGGGAGATCATCTCTGGGTGG
CCCGTTTCATCTTGCATCGTGTGTGTGAAGACTTTGGAGTGATAGCAACCTTTGATCCTA
AGCCCATTCCTGGGAACTGGAATGGTGCAGGCTGCCATACCAACTTCAGCACCAAGGCCA
TGCGGGAGGAGAATGGTCTGAAGTACATCGAGGAGGCCATTGAAACTAAGCAAGCGGC
ACCAGTACCACATCCGTGCCTATGATCAAGGGAGGCCTGGACAATGCCCGACGTCTAA
CTGGATTCCATGAAACCTCCAACATCAACGACTTTTCTGGTGGTGTAGCCAATCGTAGCG
CCAGCATACGCATTCCCCGGACTGTTGGCCAGGAGAAGAAGGGTTACTTTGAAGATCGTC
GCCCCTCTGCCAACTGCGACCCCTTTTCGGTGACAGAAGCCCTCATCCGCACGTGTCTTC
TCAATGAAACCGGCGATGAGCCCTTCCAGTACAAAAATTAAGTGGACTAGACCTCCAGCT
GTTGAGCCCCTCCTAGTTCTTCATCCCACTCCAACTCTTCCCCCTCTCCCAGTTGTCCCG
ATTGTAACTCAAAGGGTGGAATATCAAGGTCGTTTTTTTTCATTCC
SEQ ID NO: 4: GS protein grown in bacteria used in Example 1.
MGSSHHHHHHGGGGSMTTSASSHLNKGIKQVYMSLPQGEKVQAMYIWIDGTGEGLRCKTRTLDSEPKCVEELPEWNFDGSSTLQSEGSNSDMYLVPAAMFRDPFRKDPNKLVLCEVFKYNRRPAETNLRHTCKRIMDMVSNQHPWFGMEQEYTLMGTDGHPFGWPSNGFPGPQGPYYCGVGADRAYGRDIVEAHYRACLYAGVKIAGTNAEVMPAQWEFQIGPCEGISMGDHLWVARFILHRVCEDFGVIATFDPKPIPGNWNGAGCHTNFSTKAMREENGLKYIEEAIEKLSKRHQYHIRAYDPKGGLDNARRLTGFHETSNINDFSAGVANRSASIRIPRTVGQEKKGYFEDRRPSANCDPFSVTEALIRTCLLNETG DEPFQYKN
SEQ ID NO: 5 cDNA (Bacterial optimized cDNA used in Example 1).

SEQ ID NO: 6 [Lactobacillus acidophilus strain 30SC GS]
> Tr | F0TG87 | F0TG87_LACA3 Glutamine Synthetase OS = Lactobacillus acidophilus (Strain 30SC)
MSKQYTTEEIRKEVADKDVRFLRLCFTDINGTEKAVEVPTSQLDKVLTNDIRFDGSSIDGFVRLEESDMVLYPDFSTWSVLPWGDEHGGKIGRLICSVHMTDGKPFAGDPRNNLKRVLGEMKEAGFDTFDIGFEMEFHLFKLDENGNWTTEVPDHASYFDMTSDDEGARCRREIVETLEEIGFEVEAAHHEVGDGQQEIDFRFDDALTTADRCQTFKMVARHIARKHGLFATFMAKPVEGQAGNGMHNNMSLFKNKHNVFYDKDGEFHLSNTALYFLNGILEHARAITAIGNPTVNSYKRLIPGFEAPVYIAWAAKNRSPLVRIPSAGEINTRLEMRSADPTANPYLLLAACLTAGLKGIKEQKMPMKPVEENIFEMTEEERAEHGIKPLPTTLHNAIKAFKEDDLIKSALGEHLTHSFIESKELEWSKYSQSVSDWERQRYMNW
SEQ ID NO: 7 [There Myth GS] [corn / Maize GS]
> Tr | B4G1P1 | B4G1P1_MAIZE Glutamine Synthetase
MACLTDLVNLNLSDNTEKIIAEYIWIGGSGMDLRSKARTLSGPVTDPSKLPKWNYDGSSTGQAPGEDSEVILYPQAIFKDPFRRGNNILVMCDCYTPAGEPIPTNKRYNAAKIFSSPEVAAEEPWYGIEQEYTLLQKDTNWPLGWPIGGFPGPQGPYYCGIGAEKSFGRDIVDAHYKACLYAGINISGINGEVMPGQWEFQVGPSVGISSGDQVWVARYILERITEIAGVVVTFDPKPIPGDWNGAGAHTNYSTESMRKEGGYEVIKAAIEKLKLRHREHIAAYGEGNERRLTGRHETADINTFSWGVANRGASVRVGRETEQNGKGYFEDRRPASNMDPYVVTSMIAETTIIWKP

Claims (35)

Glutamine synthase (GS) for use in methods of treating or preventing fatty liver disease and related liver diseases including hepatitis, fibrosis, cirrhosis and cancer. GS for use in the method of treating or preventing fatty liver disease according to claim 1, wherein the GS reduces or eliminates hepatic fat deposition and / or improves hepatocyte or tissue fibrosis. GS for use in a method of treating or preventing fatty liver disease in a subject according to claim 1 or 2, wherein the GS is a protein or is provided as a nucleic acid molecule encoding a GS protein. GS for use in a method for treating or preventing fatty liver disease in the subject according to any one of claims 1 to 3, which is intended to be used in combination with an ammonia lowering agent. Ammonia lowering agent for use in combination with GS for use in the treatment or prevention of fatty liver disease in a subject. The treatment or prevention of fatty liver disease according to any one of claims 1 to 5, wherein the fatty liver disease is acquired fatty liver disease, for example, NAFLD, NASH, fibrosis, acute liver failure, liver cirrhosis or liver cancer. An ammonia-lowering agent combined with GS for use in the method of treatment, or GS for use in the treatment or prevention of fatty liver disease in a subject. Any of claims 1-6, wherein the fatty liver disease is genetically derived, preferably the fatty liver disease is caused by urea cycle dysfunction (UCD), organic academia and / or glutamine synthase deficiency. The GS for use in the method for treating or preventing fatty liver disease, or the ammonia lowering agent combined with GS for use in the treatment or prevention of fatty liver disease in a subject according to item 1. 1 A GS that may be combined with an ammonia-lowering agent, or an ammonia-lowering agent in combination with GS, for use in the treatment or prevention of fatty liver disease in the subject according to any one of 7 to 7. The fat in any one of claims 1-8, wherein the subject has one or more conditions selected from the group consisting of diabetes, obesity, malnutrition, excessive alcohol intake, and drug use. GS for use in the treatment or prevention of liver disease, or an ammonia-lowering agent in combination with GS. GS is a protein and contains, or is an enzymatically active fragment thereof, an amino acid sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 1; or GS is a nucleic acid molecule encoding a GS protein or The biologically active fragment or variant thereof, provided in an expression vector, for use in the method of treating or preventing adipose liver disease in the subject according to any one of claims 1-9. GS. The subject of any one of claims 1-10, wherein the GS protein is linked to a moiety selected from, for example, a protein, peptide, non-protein polymer or affinity tag, preferably the moiety is polyethylene glycol (PEG). GS for use in the treatment or prevention of fatty liver disease in, or an ammonia lowering agent in combination with GS. The subject of any one of claims 1-11, wherein the PEG is ligated to the GS protein at the N-terminus of the protein, preferably the GS protein is ligated to the moiety via a peptide linker or chemical bond. A GS for use in the treatment or prevention of fatty liver disease, or an ammonia-lowering agent in combination with the GS. The fat in the subject of any one of claims 1-12 for use according to any one of claims 8-12, wherein the GS protein is covalently linked to the moiety. GS for use in the treatment or prevention of liver disease, or an ammonia-lowering agent in combination with GS. To be used in the treatment or prevention of fatty liver disease in a subject according to any one of claims 1 to 13, wherein the GS protein is in a form suitable for systemic administration, preferably subcutaneous or intravenous administration to the subject. GS, or an ammonia lowering agent combined with GS. GS is combined with GS, or GS, for use in the treatment or prevention of fatty liver disease in the subject according to any one of claims 1-14, provided as a preparation comprising a multimeric form of protein. Ammonia lowering agent. A GS, or an ammonia-lowering agent combined with GS, for use in the treatment or prevention of fatty liver disease in the subject according to any one of claims 1 to 15, wherein the GS protein is provided in a monomeric form. Ammonia-lowering agents are engineered micros that remove nitrogen trapping agents (eg, sodium phenylacetate), ion-exchange resins (eg, Relypsa), ammonia absorbers (such as liposome-based ammonia absorbers, eg Versantis), ammonia. Fatty liver disease in the subject according to any one of claims 2-16, selected from the group consisting of intestinal ammonia detoxification methods such as biome (eg Synlogic) or the use of antibiotics (eg refaxmin) and lactulose. GS for use in the treatment or prevention of, or an ammonia-lowering agent in combination with GS. The ammonia-lowering agent is a nitrogen scavenger, and the nitrogen scavenger is a pharmaceutically acceptable salt of phenylacetic acid or a pharmaceutically acceptable prodrug thereof, a pharmaceutically acceptable salt of phenylbutyric acid, the pharmaceutical thereof. From the group consisting of an acceptable prodrug, glycerol phenylbutyric acid, a pharmaceutically acceptable prodrug thereof, a pharmaceutically acceptable salt of benzoic acid or a pharmaceutically acceptable prodrug thereof, and an ammonia binding resin. GS, or an ammonia-lowering agent in combination with GS, to be selected for use in the treatment or prevention of fatty liver disease in the subject of claim 16. GS, or a nitrogen scavenger in combination with GS, for use in the treatment or prevention of fatty liver disease in the subject of claim 18, wherein the pharmaceutically acceptable salt of phenylacetic acid is sodium phenylacetate. A kit containing a GS and an ammonia lowering agent or an amino acid thereof or a urea cycle intermediate or an analog thereof for use in a method for treating or preventing fatty liver disease in a subject. Ammonia lowering agents include nitrogen scavengers, ion exchange resins (eg Relapsa), ammonia absorbers (eg Versantis, such as liposome-based ammonia absorbers), engineered microbiomes for removing ammonia (eg Synlogic), refaxmin. 20. The kit of claim 20, selected from the group consisting of and lacturose. 21. The kit of claim 21, wherein the nitrogen scavenger is as defined in claim 18. The kit according to any one of claims 20 to 22, wherein the fatty liver disease is as defined in claims 6 to 8. The kit according to any one of claims 20 to 23, wherein the GS is as defined in any one of claims 3 or 9-13. A method for treating fatty liver disease in a subject by reducing or eliminating hepatic fat deposition and / or fibrosis, comprising administering GS to the subject. 25. The method of claim 25, further comprising administering an ammonia lowering agent. 25. The method of claim 25 or 26, comprising simultaneous, continuous or subsequent administration of glutamine synthetase (GS) and an ammonia lowering agent to the subject. The method of any one of claims 25-27, wherein the subject has or has been previously diagnosed with fatty liver disease. The method of any one of claims 25-28, wherein the GS is as defined in any one of claims 3 or 10-16. The method according to any one of claims 25 to 29, wherein the fatty liver disease is as defined in any one of claims 6 to 8. The method according to any one of claims 25 to 30, wherein the object is as defined in claim 9. The method of any one of claims 26-31, wherein the ammonia-lowering agent is as defined in any one of claims 17-19. GS provided as a composition for use in the method of treating fatty liver disease. GS for use in the method of claim 33, wherein the composition is a pharmaceutical product or supplement. Expression vectors encoding glutamine synthases or biologically active fragments or variants thereof for use in the treatment of fatty liver disease, and in combination with ammonia lowering agents for use in the treatment of fatty liver disease. A cell containing an expression vector selected from the group consisting of an expression vector encoding a glutamine synthase or a biologically active fragment or variant thereof for use.

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